Pharmacokinetic and pharmacodynamic modeling of erythropoietin administration

ABSTRACT

The present invention relates to systems and methods for obtaining optimized EPO dosage regimens for a desired pharmacodynamic/pharmacokinetic response. The system includes choosing one or more EPO dosage regimens, then using a PK/PD model to determine the pharmacodynamic/pharmacokinetic profile of one or more EPO dosage regimens, and finally selecting one of the EPO dosage regimens for administration to achieve the desired pharmacodynamic/pharmacodynamic response based on the EPO profile.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional applicationSerial No. 60/133,418, filed May 11, 1999, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to systems and methods forobtaining optimized EPO dosage regimens for a desiredpharmacodynamic/pharmacokinetic response.

BACKGROUND OF THE INVENTION

[0003] Erythropoietin (EPO) is the principal factor responsible for theregulation of red blood cell production during steady-state conditionsand for accelerating recovery of red blood cell mass followinghemorrhage. EPO is a glycoprotein hormone with a molecular mass of 30KDa and is heavily glycosylated, which serves to protect the EPOmolecule from rapid degradation in vivo. Serum EPO concentrations inhumans normally range from 6 to 32 U/I (1), and the half-life (t_(1/2))of EPO is reported to range from 2 to 13 hours with a volume ofdistribution close to plasma volume. As expected for a largesialoglycoprotein, less than 10% of EPO is excreted in the urine (see,e.g., Lappin et al., 1996. Clin. Lab Haem. 18:137-145.)

[0004] The primary site for EPO synthesis in adult organisms is thekidney; although the liver and bone marrow have also been implicated,the data remains inconclusive. The primary stimulus for increased EPOsynthesis is tissue hypoxia, which results from decreased oxygenavailability in the tissues. Hypoxia can result from the loss of largeamounts of blood, destruction of red blood cells by radiation, orexposure to high altitudes. In addition, various forms of anemia causehypoxia since red blood cells are responsible for oxygen transport inthe body. In the normal state, an increased level of EPO stimulates theproduction of new red blood cells thereby raising the level of oxygenand reducing or eliminating the hypoxic condition.

[0005] The principal function of EPO is to act synergistically withother growth factors to stimulate the proliferation and differentiationof erythrocytic progenitor cells in the bone marrow leading toreticulocytosis and increased RBC numbers in the blood, a process alsoknown as erythropoiesis (FIG. 1). During erythropoiesis, celldifferentiation along the erythroid lineage occurs over a two week spanin humans. The earliest progenitor is the BFU-E (Burst-FormingUnit-Erythroid), which is small and without distinguishing histologiccharacteristics. The stage after the BFU-E is the CFU-E (Colony FormingUnit-Erythroid), which is larger than the BFU-E and immediately precedesthe stage where hemoglobin production begins. The cells that beginproducing hemoglobin are the immature erythrocytes, which not only beginproducing hemoglobin, but also start condensing their nuclei toeventually become mature erythroblasts. The mature erythroblasts aresmaller than the immature erythrocytes and have a tightly compactednucleus, which is expelled as the cells become reticulocytes.Reticulocytes are so named because these cells contain reticularnetworks of polyribosomes and as the reticulocytes lose theirpolyribosomes, they become mature red blood cells (RBCs).

[0006] Until recently, the availability of EPO has been very limited.Although the protein is present in human urine, excreted levels are toolow to make this a practical source of EPO for therapeutic uses. Theidentification, cloning, expression of genes encoding EPO and EPOpurification techniques, e.g., as described in U.S. Pat. Nos. 4,703,008,5,389,541, 5,441,868, 5,614,184, 5,688,679, 5,888,774, 5,888,772, and5,856,298, has made EPO readily available for therapeutic applications.A description of the purification of recombinant EPO (rHuEPO) from cellmedium that supported the growth of mammalian cells containingrecombinant EPO plasmids for example, is included in U.S. Pat. No.4,667,016. This recombinant EPO has an amino acid sequence identical tothat of human urinary erythropoietin, and the two are indistinguishablein chemical, physical and immunological tests. The expression andrecovery of biologically active recombinant EPO from mammalian cellhosts containing the EPO gene on recombinant plasmids has made availablequantities of EPO suitable for therapeutic applications. In addition,knowledge of the gene sequence and the availability of larger quantitiesof purified protein has led to a better understanding of the mode ofaction of this protein.

[0007] The biological activity of a protein is dependent upon itsstructure. In particular, the primary structure of a protein, i.e., itsamino acid sequence, provides information that allows the formation ofsecondary (e.g., α-helix or β-pleated sheet) and tertiary (overall3-dimensional folding) structures by a polypeptide during and aftersynthesis. Furthermore, not only is the biological activity of a proteingoverned by its structure, but also by modifications generated after theprotein has been translated. Indeed, many cell surface proteins andsecretory proteins are modified by one or more oligosacchride groups.This modification known as glycosylation, can dramatically affect thephysical properties of proteins and can be important in proteinstability, secretion, and subcellular localization. Proper glycosylationcan be essential for biological activity.

[0008] Both human urinary derived and recombinant EPO (expressed inmammalian cells) having the amino acid sequence 1-165 of human EPOcontain three N-linked and one O-linked oligosacchride chains whichtogether comprise about 40% of the total molecular weight of theglycoprotein. The oligosacchride chains have been shown to be modifiedwith terminal sialic acid residues. Enzymatic treatment of glycosylatedEPO to remove all sialic acid residues results in a loss of in vivoactivity, but does not affect its in vitro activity (Lowy et al., 1960,Nature 185:102; Goldwasser et al., 1974, J. Biol. Chem. 249:4202). Thisbehavior has been explained by rapid clearance of asialoerythropoeitinfrom the circulation upon interaction with the hepaticasialoglycoprotein binding protein (Morrell et al., 1968, J. Biol. Chem.243:155; Briggs et al., 1974, Am. J. Physiol. 227:1385; and Ashwell etal., 1978 Methods of Enzymol. 50:287). Thus, EPO possesses in vivobiological activity only when it is sialylated to avoid binding by thehepatic binding protein.

[0009] Deficient (or inefficient) EPO production relative to hemoglobinlevel is associated with certain forms of anemia. These include anemiaof renal failure and end-stage renal disease, anemia of chronicdisorders (chronic infections and rheumatoid arthritis), autoimmunedisease, acquired immune deficiency disease (AIDS), and malignancy. Manyof these conditions are associated with the generation of a factor thathas been shown to be an inhibitor of EPO activity. Other anemias areclearly EPO-independent, and include aplastic anemia, iron deficiencyanemia, the thalassemias, megaloblastic anemia, pure red cell aplasia,and myelodysplastic syndromes.

[0010] The measurement of EPO levels in human serum has clinicalimportance. Determination of EPO levels in patient serum can be usefulin distinguishing those anemias and polycythemias that are associatedwith decreased or increased EPO levels from those that are not.Additionally, the demonstration of an inappropriately low level of serumEPO is a prerequisite for concluding that an anemic patient may benefitfrom treatment with exogenous EPO.

[0011] In clinical trials, Epoetin alfa has been evaluated in normalpatients as well as in patients with various anemic conditions. Epoetinalfa induces a brisk haematological response in normal human volunteers,provided that adequate supplies of iron are available to supportincreased hemoglobin synthesis. A majority of trials have investigatedthe safety and effectiveness in the treatment of anemia associated withrenal failure. In addition, Epoetin alfa may be used to correct anemiain other patient groups including anemia associated with platinum-basedcancer chemotherapy, anemia associated with zidovudine therapy inpatients with AIDS, and anemia associated with other drugs such ascisplatin. Also, the administration of Epoetin alfa has many otherpotential therapeutic applications: Epoetin alfa administrationincreases the capacity for autologous blood donation in patientsscheduled to undergo surgery and attenuates the decrease in hemocritoften seen in untreated autologous donors; Epoetin alfa administrationincreases red blood cell recovery after allogeneic—but notautologous—bone marrow transplant; and administration of Epoetin alfahas been shown to improve the quality of life in individuals afflictedwith rheumatoid arthritis An alternative application of EPO is forenhancing the performance of athletes by causing an increase in thehematocrit of the athlete. This augmentation in hematocrit increases thecapacity of oxygen transported from the lungs to the exercising skeletalmuscles. Since the synthesis of EPO by bioengineering, injectingathletes with EPO, also known as blood doping, has become popular insports in general, and in particular, cycling (Scheen, A J., 1998. Rev.Med. Liege 53(8): 499-502).

[0012] Presently, there are a number of disadvantages associated as thestandard EPO dosage regimen administered to patients. In specificindications, such as cancer, subjects are treated with 150 IU/kg of EPOthree times per week. Thus, it remains an important goal to change thecurrently approved dosing schedule to a more convenient dosing scheduleand regimen. It is expected that a less frequent administration willimprove user acceptance and convenience. Moreover, the standard dosingregimens may not maximize the patient's physiological response; andstandard dosing regimens may not be the most cost efficient.

[0013] Furthermore, there are a number of disadvantages associated withthe route of EPO administration: regular intravenous administration isinconvenient for the patient; intravenous administration is impracticalfor individuals afflicted with certain conditions such as continuousambulatory peritoneal dialysis and non-dialysis patients with restrictedvascular access; the rapid dose delivery of rHuEPO via intravenousadministration results in a lower bioavailability of rHuEPO for longertime periods and may not be as effective for stimulating production ofRBC as desired.

[0014] Hence, for all of the reasons detailed above, a better route ofadministration and means for determining an effective dose and dosageregimen for EPO administration is needed.

[0015] Therefore, one aspect of the present invention is the developmentof a pharmacokinetic/pharmacodynamic (PK/PD) model for characterizingand predicting responses to rHuEPO thereby identifying the mostefficient, cost effective, and/or convenient treatment regimens forpatients. In a particular embodiment of the present invention,once-weekly or once every two weeks EPO administration is contemplated.Another aspect of the present invention provides a methodology toevaluate the pharmacokinetic and pharmacodynamic profiles of EPO afteradministration of two or more dosing regimens for comparison of clinicaloutcomes along with tolerance and safety parameters between the EPOdosing regimens. Associated business methods and computer systems arealso contemplated.

SUMMARY OF THE INVENTION

[0016] A specific embodiment of the present invention may include amethod for obtaining optimized EPO dosage regimens for a desiredpharmacodynamic response, which can comprise choosing one or more EPOdosage regimens, then using a PK/PD model to determine thepharmacodynamic profile of one or more EPO dosage regimens, and finallyselecting one of the EPO dosage regimens for administration to achievethe desired pharmacodynamic (PD) response based on the EPO profile. Inan additional embodiment, the PD response can comprise one or more ofthe group consisting of reticulocyte number, RBC number, and hemoglobinlevel.

[0017] An alternate embodiment of the present invention may also be amethod for obtaining optimized EPO dosage regiments for a desiredpharmacodynamic response which comprises first selecting one or moredesired pharmacodynamic responses, then using a PK/PD model to determinea EPO dosage regimen that provides the desired responses, and finally,selecting one of the EPO dosage regimens for administration to achievethe desired pharmacodynamic response. In an additional embodiment, thePD response can comprise one or more of the group consisting ofreticulocyte number, RBC number, and hemoglobin level.

[0018] An additional preferred embodiment of the present invention caninclude a computer program, which can be used for obtaining optimizeddosage regimens for a desired pharmacodynamic response. The computerprogram may comprise a computer code. In a further embodiment, thecomputer code describes a PK/PD model for EPO and allows the user toselect one or more desired pharmacodynamic responses. The computer codethen uses the PK/PD model to determine EPO dosage regimens that wouldprovide the desired pharmacodynamic responses. The EPO dosage regimenmay be administered as a weekly or once every two weeks, based upon bodymass, dose. Preferably, the weekly EPO dose may comprise administeringEPO at a dosing of 40,000 IU/kg and the once every two weeks EPO dosingregimen may comprise administration of EPO at a dosing of about 80,000to about 120,000 IU/kg. In an additional embodiment, the PD response cancomprise one or more of the group consisting of reticulocyte number, RBCnumber, and hemoglobin level.

[0019] An alternate preferred embodiment of the present invention mayinclude a computer program for obtaining optimized dosage regimens for adesired pharmacodynamic response. In an additional embodiment, thecomputer program comprises a computer code. The computer code may allowthe user to select one or more EPO dosage regimens. The computer codethen uses the PK/PD model to determine a pharmacodynamic response basedon the EPO dosage regimens selected.

[0020] A preferred embodiment of the present invention may includecomputer program for determining optimized EPO dosage regimens for adesired pharmacokinetic response comprising the steps of choosing one ormore EPO dosage regimens, using the PK/PD model to determine thepharmacokinetic response of the EPO dosage regimens, and then selectingthe desired EPO dosage regimen based on pharmacokinetic profile, in aspecific embodiment, based upon one ore more EPO or EPO-like compounds.In an additional embodiment, the pharmacokinetic response may includeserum EPO levels, bioavailability, and EPO threshold levels.

[0021] A further embodiment of the present invention may include amethod for obtaining optimized EPO dosage regimens for a desiredpharmacokinetic response comprising the steps of first selecting one ormore desired pharmacokinetic responses, then using a PK/PD model todetermine a EPO dosage regimen that provides one or more of the desiredpharmacokinetic responses, and finally selecting the EPO dosage regimenthat provides the desired pharmacokinetic responses.

[0022] An additional embodiment of the present invention can include acomputer program for obtaining optimized EPO dosage regimens for adesired pharmacokinetic response which comprises a computer code thatdescribes a PK/PD model for EPO. In a further embodiment, the computercode may allow the user to select of one or more pharmacokineticresponses, and then use the PK/PD model to determine one or more EPOdosage regimens that provide the desired pharmacokinetic responses.

[0023] An alternate preferred embodiment of the present invention mayinclude a computer program for obtaining optimized dosage regimens for adesired pharmacokinetic response. In an additional embodiment, thecomputer program comprises a computer code. The computer code may allowthe user to select one or more EPO dosage regimens. The computer codethen uses the PK/PD model to determine a pharmacokinetic response basedon the EPO dosage regimens selected.

[0024] One or more EPO or EPO-like compounds may be contemplated foruse. Another preferred embodiment of the present invention comprises avariety of methods including a business method of providing to aconsumer an EPO dosing regimen that comprises a first dose of EPOfollowed by a second dose of EPO to a patient. The second dose of EPO ispreferably administered to the patient at a time point after the firstdose that coincides with the PD profile resulting from the first dose ofEPO. The PD profile may include, number of progenitor cells produced inrespect to time, reticulocyte concentration in respect to time, RBCnumber produced in respect to time, and hemoglobin concentration inrespect to time. Most preferably, the PD profile will be thereticulocyte profile for this regimen. The second dose of EPO ispreferably administered to coincide with the reticulocyte profile, i.e.,when reticulocyte production peaks. The second dose of EPO facilitatesthe maturation of young red cells in the circulation into mature RBCs.

[0025] A further embodiment of the present invention comprises abusiness method of providing to a patient an EPO dosing regimen thatcomprises a first dose of EPO followed by a second dose of EPO to apatient. The second dose of EPO is administered to the patient at a timeafter the first dose that coincides with the reticulocyte profile of thepatient. The second dose may be administered within 6 to 10 daysfollowing the first EPO dose. Preferably, the second EPO will beadministered 7 days subsequent to the first EPO dose.

[0026] The business method of the present application relates to thecommercial and other uses, of the methodologies of the presentinvention. In one aspect, the business method includes the marketing,sale, or licensing of the present methodologies in the context ofproviding consumers, i.e., patients, medical practitioners, medicalservice providers, and pharmaceutical distributors and manufacturers,with the EPO dosing regimens provided by the present invention. Theseinclude once weekly and once every two weeks EPO dosing regimens.

[0027] Another preferred embodiment of the present invention provides amethod for creating a pharmacokinetic model for subcutaneous (SC) EPOadministration in patients. This method can comprise obtainingpharmacokinetic data from patients, choosing an equation based the PKdata collected from the patients, and fitting the PK data to theequation. In addition, obtaining the PK data may comprise normalizingserum EPO concentration values from the collected PK data and creatingserum EPO concentration time profiles based on the normalized data. In afurther embodiment, the PK data may be normalized by first obtainingbaseline serum EPO concentration values from the PK data by averagingpredose serum EPO concentration values at a plurality of time points;next, obtaining serum EPO concentration values following SC EPOadministration; then, obtaining normalized serum EPO concentrationvalues by subtracting predose EPO concentration values from serum EPOconcentration values; and, finally, calculating mean normalized serumEPO concentration values at each time point.

[0028] In an additional embodiment of the present invention, the PKequation may comprise selecting the Michaelis-Menten equation. The PKdata may be fitted to the PK equation using, for example, ADAPT IIsoftware and an estimate of parameters may be obtained by utilizing theleast-squares by Maximum likelihood method and the extended leastsquares model. In a further embodiment, the parameters may be selectedfrom the group consisting of Vmax, Km, Vd, Fr, τ (lower doses), and τ(higher doses).

[0029] A further embodiment of the present invention provides a methodfor calculating the bioavailability of EPO following SC EPOadministration. The method may comprise obtaining PK data, calculatingthe area under the serum EPO concentration curve (AUC) versus dose,normalizing AUC to dose, and finally, deriving an equation by performinga linear regression of the PK data.

[0030] Another preferred embodiment of the present invention provides amethod for creating a pharmacodynamic (PD) model after SC EPOadministration. This method may comprise normalizing serum EPOconcentrations, obtaining PD data, choosing a PD model, obtaining anequation based on the PD model, and fitting the PD data to the PDequations. In an additional embodiment, normalizing the serum EPOconcentrations may comprise obtaining baseline serum EPO concentration(Cbs) for each dose group by averaging predose serum EPO concentrationvalues at a plurality of time points for each dose group, and then,adjusting C_(bs) by adding Cb_(bs) to serum EPO concentration predictedby PK model and where the adjusted C_(bs) can be used as a forcingfunction for PD analysis.

[0031] In a further embodiment, the PD data may be obtained bydetermining the mean predose precursor cell, reticulocyte, and RBCnumber, and hemoglobin concentration, and then obtaining meanreticulocyte-, mean RBC-, and mean hemoglobin-versus time profilesaccording to EPO dose.

[0032] In an additional embodiment, the PD model may comprise a cellloss and production model. The PD data may be fitted to the modelequation by using, for example, ADAPT II software, and following, bothestimate and fixed parameters may be obtained by utilizing theleast-squares by Maximum likelihood method and extended least squaresmodel. Additionally, the estimated parameters can comprise Ks, SC₅₀, andTP, while the fixed parameters may include R_(L), RBC_(L), Hb, andthreshold.

[0033] A further preferred embodiment of the present invention mayprovide a method for predicting a PD response in a patient followingvarious doses of SC EPO. Moreover, this method may comprise selecting adose and dosage regimen, and then determining the PD response based onthat particular dose and dosage regimen via the PK/PD model. In anadditional embodiment, the PD response can comprise one or more of thegroup consisting of reticulocyte number, RBC number, and hemoglobinlevel.

[0034] The present invention can address the requirements of patientsthat may have deficient or inefficient EPO production relative tohemoglobin level, which may be associated with certain forms of anemia.These may include, but are not limited to, anemia associated withend-stage renal or renal failure related anemia, platinum based cancerchemotherapy related anemia, AIDS drug therapy related anemia where thedrugs may include cisplatin and zidovudine. Also, patients may beundergoing autologous transfusion prior to surgery, recovering from anallogenic bone marrow transplant, suffering from rheumatoid arthritis,or an athlete or others requiring or desiring increased RBC numbersand/or hemoglobin.

[0035] The PK/PD model of the present invention has many potentialtherapeutic applications. For example, a physician can use this PK/PDmodeling system to determine the optimal EPO dosage regimen toadminister to a patient in need of increased RBC numbers and/orhemoglobin. In particular, the physician would have the option of eitherdetermining an EPO dosage regimen based on the desired pharmacodynamicoutcome or determining a pharmacodynamic response based on a specificEPO dosage regimen.

BRIEF DESCRIPTION OF THE FIGURES

[0036]FIG. 1: Process of Erythropoiesis.

[0037]FIG. 2: Serum rHuEPO concentration versus time profiles afterintravenous administration of five indicated dose levels. Data for the150 and 300 IU/kg doses are the mean data from six healthy subjectswhile the other doses are single subject data. Circles are the datacorrected for the baseline EPO concentrations while the solid lines areobtained from fitting the data to equations 1, 2 and 3, infra.

[0038]FIG. 3: Pharmacokinetic parameters for intravenous andsubcutaneous EPO doses.

[0039]FIG. 4: Schematic representation of a pharmacokinetic model of thepresent invention used for analysis of plasma rHuEPO (C_(EPO)) versustime profiles. The symbols used are defined in the definitions sectionof the detailed description of the invention, infra.

[0040]FIG. 5A: Serum rHuEPO concentration versus time profiles aftersubcutaneous administration of 300, 450, 600, and 900 IU/kg doses. Datapoints for each dose are the mean values of five healthy subjects. Thedata are corrected for baseline EPO concentrations while the solid lineis obtained from fitting the data to equations 1, 2 and 3.

[0041]FIG. 5B: Serum rHuEPO concentration versus time profiles aftersubcutaneous administration of 1200, 1350, 1800, and 2400 IU/kg doses.Data points for each dose are the mean values of five healthy subjects.The data are corrected for baseline EPO concentrations while the solidline is obtained from fitting the data to equations 1, 2 and 3.

[0042]FIG. 6: Area under the serum rHuEPO concentration-time curve (AUC)versus dose after subcutaneous administration of the eight dose levelsindicated in FIGS. 4A and 4B. The AUC was calculated by the Splinemethod.

[0043]FIG. 7: Bioavailability (F) of the rHuEPO versus dose aftersubcutaneous administration of the eight indicated dose levels. The Fvalues were obtained from the initial fittings of the pharmacokineticdata to the model as explained in the text. Linear regression yielded ar² of 0.9713, slope of 0.00024952, and an intercept of 0.3884.

[0044]FIG. 8: Bioavailability values for subcutaneous rHuEPOadministration.

[0045]FIG. 9: Serum rHuEPO concentration versus time profiles duringmultiple-dosing regimens of 150 IU/kg t.i.w. (top) and 600 IU/kg/week(bottom). Solid circles represent mean data while lines aremodel-predicted values.

[0046]FIG. 10: Schematic representation of the pharmacodynamic modelused for analysis of reticulocyte, RBC, and hemoglobin concentrations.Symbols are defined in the definition section of the detaileddescription of the present invention, infra.

[0047]FIG. 11: Mean reticulocyte count versus time profiles for theeight indicated subcutaneous rHuEPO dose levels.

[0048]FIG. 12A: Reticulocyte number versus time profiles aftersubcutaneous administration of 300, 450, 600, and 900 IU/kg doses. Datafor each dose are mean values from five healthy subjects. Symbolsindicate the experimental data while the solid lines were obtained fromfitting the data to equations 4, 5, 6, and 7, infra.

[0049]FIG. 12B: Reticulocyte number versus time profiles aftersubcutaneous administration of 1200, 1350, 1800, and 2400 IU/kg doses.Data for each dose are mean values from five healthy subjects. Symbolsare the experimental data while the solid lines were obtained fromfitting the data to equations 4, 5, 6 and 7.

[0050]FIG. 13: Estimated and fixed pharmacodynamic parameters forsubcutaneous EPO effects.

[0051]FIG. 14: Hemoglobin concentration versus time profiles aftersingle subcutaneous administration of the eight indicated dose levels ofrHuEPO. Closed circles are the mean data while solid lines are the modelpredictions.

[0052]FIG. 15: Reticulocyte, RBC, and hemoglobin responses aftermultiple subcutaneous dosing of 150 IU/kg t.i.w. rHuEPO. Solid circlesrepresent measured data and the solid lines are the model-predictions.

[0053]FIG. 16: Reticulocyte, RBC, and hemoglobin responses aftermultiple dosing of 600 IU/kg/week rHuEPO. Solid circles representmeasured data and the solid lines are the model-predictions.

[0054]FIG. 17: Summary of Epoetin Alfa clinical pharmacokinetic studiescontributing to pharmacokinetic and pharmacodynamic data for subjects inClinical Studies EPO-PHI-358, EPO-PHI-359, EPO-PHI-370, and EPO-PHI-373.

[0055]FIG. 18A: Biopharmaceutics study summary for Clinical StudyEPO-PHI-373.

[0056]FIG. 18B: Biopharmaceutics study summary for Clinical StudyEPO-PHI-370.

[0057]FIG. 18C: Biopharmaceutics study summary for Clinical StudyEPO-PHI-358.

[0058]FIG. 18D: Biopharmaceutics study summary for Clinical StudyEPO-PHI-359.

[0059]FIG. 19: Summary of pharmacokinetic data for Clinical StudiesEPO-PHI-358, EPO-PHI-359, EPO-PHI-370, and EPO-PHI-373.

[0060]FIG. 20: Summary of analytical methods for Clinical StudiesEPO-PHI-358, EPO-PHI-359, EPO-PHI-370, and EPO-PHI-373.

[0061]FIG. 21: Mean±SD demographic and baseline parameters for subjectsenrolled in Clinical Studies EPO-PHI-358 and EPO-PHI-359.

[0062]FIG. 22: Mean serum Epoetin Alfa concentration-Time profiles(uncorrected for baseline EPO) for subjects in Clinical StudyEPO-PHI-358.

[0063]FIG. 23: Mean serum Epoetin Alfa concentration-Time profiles(uncorrected for baseline EPO) for subjects in Clinical StudyEPO-PHI-359

[0064]FIG. 24: Mean±SD (% CV) pharmacokinetic parameters (ClinicalStudies EPO-PHI-358 and EPO-PHI-359).

[0065]FIG. 25: Relationship between mean±SD C_(max) and dose forsubjects receiving single or multiple SC dose regimens in ClinicalStudies EPO-PHI-358 and EPO-PHI-359.

[0066]FIG. 26: Relationship between mean±SD CL/F and dose for subjectsreceiving single or multiple SC dose regimens in Clinical StudiesEPO-PHI-358 and EPO-PHI-359.

[0067]FIG. 27: Mean percent reticulocyte-time profiles for the four weekstudy period (Clinical Studies EPO-PHI-358 and EPO-PHI-359).

[0068]FIG. 28: Mean±SC change in hemoglobin from baseline profiles after150 IU/kg t.i.w. (N=5) and 600 IU/kg q.w (N=5) Epoetin Alfa for fourweeks (Clinical Studies EPO-PHI-358 and EPO-PHI-359).

[0069]FIG. 29: Demographic data of subjects in Clinical StudyEPO-PHI-370.

[0070]FIG. 30: Mean serum concentration-time profiles of Epoetin Alfa(uncorrected for Baseline EPO) in healthy subjects after receiving 150IU/kg t.i.w. (N=24) or 40,000 IU q.w (N=22) during the fourth dosingweek (Clinical Study EPO-PHI-370).

[0071]FIG. 31: Mean±SD (% CV) pharmacokinetic parameters (Clinical StudyEPO-PHI-370).

[0072]FIG. 32: Profile of mean change from baseline in percentreticulocytes.

[0073]FIG. 33: Profile of mean change form baseline in hemoglobin(g/dl).

[0074]FIG. 34: Mean±SD (% CV) pharmacodynamic parameters corrected forbaseline value (Clinical Study EPO-PHI-370).

[0075]FIG. 35: Mean±SD demographic data of subjects in Clinical StudyEPO-PHI-373.

[0076]FIG. 36: Mean serum concentration-time profiles of Epoetin Alfa(uncorrected for Baseline EPO) in healthy subjects after receiving 150IU/kg t.i.w. (N=17) or 40,000 IU q.w (N=17) during the fourth dosingweek (Clinical Study EPO-PHI-373).

[0077]FIG. 37: Mean±SD (% CV) pharmacokinetic parameters corrected forbaseline value (Clinical Study EPO-PHI-373).

[0078]FIG. 38: Profile of mean change from baseline in percentreticulocytes for subjects in Clinical Study EPO-PHI-373.

[0079]FIG. 39: Profile of mean change from baseline in hemoglobin (g/dI)for subjects in Clinical Study EPO-PHI-373.

[0080]FIG. 40: Profile of mean change from baseline total red bloodcells (×10¹²/l) for subjects in Clinical Study EPO-PHI-373.

[0081]FIG. 41: Mean±SD (% CV) pharmacodynamic parameters corrected forbaseline value for subjects in Clinical Study EPO-PHI-373.

[0082]FIG. 42: Mean±SD (% CV) pharmacokinetic parameters corrected forbaseline value for subjects in Clinical Studies EPO-PHI-358,EPO-PHI-359, EPO-PHI-370, and EPO-PHI-373.

[0083]FIG. 43: Mean AUC of change in percent reticulocytes as a functionof AUC (Day 0-29) of Epoetin Alfa (Clinical Studies EPO-PHI-358 andEPO-PHI-359).

[0084]FIG. 44: Mean AUC of change in percent reticulocytes as a functionof AUC (Day 0-29) of Epoetin Alfa (Clinical Studies EPO-PHI-370 andEPO-PHI-373).

[0085]FIG. 45: Profile of mean change from baseline in hemoglobin (g/dl)for subjects in Clinical Study EPO-PHI-373.

[0086]FIG. 46: Profile of mean change from baseline in hemoglobin (g/dl)for subjects in Clinical Study EPO-PHI-370.

[0087]FIG. 47: Demographic and baseline characteristics of the 34subjects who completed the EPO study (EPO-PHI-373). 18 subjects werepart of Group 1 who were administered an EPO dosing regimen of 150 IU/kgt.i.w. and 18 subjects were part of Group 2 who were administered EPO at40,000 q.w. Demographic characteristics include sex, age (years), weight(kg), height (cm), and race.

[0088]FIG. 48: Serum epoetin alfa concentrations uncorrected for predoseendogenous erythropoietin concentrations for subjects in Group 1 (150IU/kg t.i.w.) designated by triangles and Group 2 (40,000 IU q.w)denoted by cirlces.

[0089]FIG. 49: Serum epoetin alfa concentration data corrected forpredose endogenous erythropoietin concentrations for subjects in Group 1(150 IU/kg t.i.w.) designated by triangles and Group 2 (40,000 IU q.w.)designated by circles.

[0090]FIG. 50: Mean (SD) [% CV] pharmacokinetic parameter values withdata for individual subjects in Group 1 (150 IU/kg .i.w.) and Group 2(40,000 IU q.w).

[0091]FIG. 51: Summaries of mean (SD) change from baseline in percentreticulocytes by study day for the efficacy population for all subjectsin dosing Groups 1 (150 IU/kg t.i.w.) and Group 2 (40,000 IU q.w) forClinical Study EPO-PHI-373.

[0092]FIG. 52: Profile of mean (SD) change from baseline in percentreticulocytes by study day for the efficacy population for all subjects.The open circles represent Group 1 (150 IU/kg t.i.w.) and the closedcircles represent Group 2 (40,000 IU q.w). The parameters obtained arelisted in FIG. 51.

[0093]FIG. 53: Summaries of mean (SD) change from baseline in hemoglobin(g/dL) by study day for the efficacy population for all subjects inGroup 1 (150 IU/kg t.i.w.) and Group 2 (40,000 IU q.w) for ClinicalStudy EPO-PHI-373.

[0094]FIG. 54: Profile of mean (SD) change from baseline in percenthemoglobin (g/dL) by study day for the efficacy population for allsubjects. The open circles represent Group 1 (150 IU/kg t.i.w.) and theclosed circles represent Group 2 (40,000 IU q.w). The parametersobtained are listed in FIG. 53.

[0095]FIG. 55: Summaries of mean (SD) change from baseline in red bloodcells (×10¹²/L) by study day for the efficacy population for allsubjects in Clinical Study EPO-PHI-373.

[0096]FIG. 56: Profile of mean (SD) change from baseline in red bloodcells (×10¹²/L) by study day for the efficacy population for allsubjects. The open circles represent Group 1 (150 IU/kg t.i.w.) and theclosed circles represent Group 2 (40,000 IU q.w). The parametersobtained are listed in FIG. 55.

[0097]FIG. 57: Mean pharmacodynamic parameter values corrected forbaseline value. % CV is the percent coefficient of variation. The notesare as follows: ^(a)AUC of % reticulocytes over the one month studyperiod and corrected for predose baseline value; ^(b)AUC of hemoglobinover one month study period and corrected for predose baseline value;^(c)AUC of red blood cells over one month study period and corrected forpredose baseline value; ^(d)ratios of 40,000 IU q.w. to 150 IU/kg t.i.w.mean parameter value for all subjects; ^(e)including all female subjectsin both treatment groups; ^(f)including all male subjects in bothtreatment groups; ^(g)statistically different (p<0.05) between male andfemale subjects.

[0098]FIG. 58: Treatment-emergent adverse events by preferred term forindividual subjects in Clinical Study EPO-PHI-373.

[0099]FIG. 59: The mean changes from baseline in iron, ferritin,transferrin saturation, and other serum chemistry parameters by bothtreatment group and study day. Group 1 was administered 150 IU/kg t.i.w.EPO and Group 2 was administered 40,000 IU/kg q.w. in Clinical StudyEPO-PHI-373.

[0100]FIG. 60: The profile of mean change from baseline in iron,ferritin (ng/mL) by both treatment group and study day for ClinicalStudy EPO-PHI-373. Group 1 (150 IU/kg t.i.w.) is designated by opencircles and Group 2 (40,000 IU/kg q.w) is designated by closed circles.The parameters obtained for ferritin are listed in FIG. 59.

[0101]FIG. 61: Summary of the mean changes from baseline in vital signmeasurements for individuals in Group 1 (150 IU/kg t.i.w.) in ClinicalStudy EPO-PHI-373.

[0102]FIG. 62: Schematic representation of the model for erythropoiesisstimulating effects of rHuEPO.

[0103]FIG. 63: Pharmacokinetic parameters after rHuEPO dosing of EPREX®and PROLEASE®.

[0104]FIG. 64: Pharmacodynamic parameters after rHuEPO dosing. Thereticulocyte data for males and females were analyzed separately. Theseparameters may reflect some slight pharmacodynamic differences basedupon gender.

[0105]FIG. 65: Profile of pharmacodynamic parameters afteradministration of 600 IU/kg/t.i.w. EPREX® for 4 weeks. The mean rHuEPOconcentration-time, reticulocyte concentration-time, and RBCconcentration-time profiles are shown. The male subjects are designatedby the closed circles while the female subjects are designated with opencircle.

[0106]FIG. 66: Profile of pharmacodynamic parameters afteradministration of 150 IU/kg/t.i.w. EPREX® for 1 month. The mean rHuEPOconcentration-time, reticulocyte concentration-time, and RBCconcentration-time profiles are shown. The male subjects are designatedby the closed circles while the female subjects are designated with opencircle.

[0107]FIG. 67: Profile of pharmacodynamic parameters afteradministration of PROLEASE® at 2400 IU/kg/mth single dose. The meanrHuEPO concentration-time, reticulocyte concentration-time, and RBCconcentration-time profiles are shown. The male subjects are designatedby the closed circles while the female subjects are designated with opencircle.

[0108]FIG. 68: Simulations of hemoglobin levels after administration ofdifferent doses/regimens of rHuEPO.

[0109]FIG. 69: Simulations of hemoglobin and RBC response versus timeprofiles for the EPO dose regimen of 600 IU/kg/wk for 24 weeks incomparison to giving a total dose of 40,000 IU/wk of rHuEPO to subjectswith body weights of 50, 70 and 90 kg.

[0110]FIG. 70A: Simulations of reticulocyte, RBC, and hemoglobinresponse versus time profiles in cancer patients for the EPO doseregimen of 150 IU/kg/t.i.w. for 12 weeks.

[0111]FIG. 70B: Simulations of reticulocyte, RBC, and hemoglobinresponse versus time profiles in cancer patients for the EPO doseregimen of 300 IU/kg/t.i.w. for 12 weeks.

[0112]FIG. 70C: Simulations of reticulocyte, RBC, and hemoglobinresponse versus time profiles in cancer patients for the EPO doseregimen of 450 IU/kg/t.i.w. for 12 weeks.

[0113]FIG. 70D: Simulations of reticulocyte, RBC, and hemoglobinresponse versus time profiles in cancer patients for the EPO doseregimen of 600 IU/kg/t.i.w. for 12 weeks.

[0114]FIG. 70E: Simulations of reticulocyte, RBC, and hemoglobinresponse versus time profiles in cancer patients for the EPO doseregimen of 900 IU/kg/t.i.w. for 12 weeks.

[0115]FIG. 71: Mean hemoglobin time concentration profiles by day forimmunosuppressed (IS) and non-immunosuppressed dogs. The closed circlesrepresent EPO administered at 50 IU/kg/d IS dogs, the closed squaresrepresent EPO administered at 50 IU/kg/d t.i.w. for non-IS dogs, theopen triangle represents EPO administered at 600 IU/kg/wk for IS dogs,the closed triangles represent EPO administered at 600 IU/kg/wk fornon-IS dogs, the closed diamonds represent saline control for IS dogs,and the shaded octagons represent the saline control for non-IS dogs.

[0116]FIG. 72: Mean red blood cell time-concentration profiles by dayfor immunosuppressed and nonimmunosuppressed dogs. The closed circlesrepresent EPO administered at 50 IU/kg/d IS dogs, the closed squaresrepresent EPO administered at 50 IU/kg/d t.i.w. for non-IS dogs, theopen triangle represents EPO administered at 600 IU/kg/wk for IS dogs,the closed triangles represent EPO administered at 600 IU/kg/wk fornon-IS dogs, the closed diamonds represent saline control for IS dogs,and the shaded octagon represent the saline control for non-IS dogs.

[0117]FIG. 73: PK/PD model for rHuEPO in monkeys.

[0118]FIG. 74: The fittings for the rHuEpo concentration-time profilesafter administrations of three single intravenous doses and six singlesubcutaneous doses of EPREX®. The parameters obtained are listed in FIG.75.

[0119]FIG. 75: PK parameters in monkeys.

[0120]FIG. 76A: Mean reticulocyte concentration-time profiles for the400 IU/kg, 1000 IU/kg, and 2400 IU/kg dosing regimens.

[0121]FIG. 76B: Mean reticulocyte concentration-time profiles for the5000 IU/kg, 20,000 IU/kg, and 40,000 IU/kg dosing regimens.

[0122]FIG. 77: PD parameters in monkeys.

[0123]FIG. 78A: Mean RBC concentration-time profiles for the 400 IU/kg,1000 IU/kg, and 2400 IU/kg dosing regimens.

[0124]FIG. 78B: Mean RBC concentration-time profiles for the 5000 IU/kg,20,000 IU/kg, and 40,000 IU/kg dosing regimens.

[0125]FIG. 79A: Mean hemoglobin concentration profiles for the 400IU/kg, 1000 IU/kg, and 2400 IU/kg dosing regimens.

[0126]FIG. 79B: Mean hemoglobin concentration profiles for the 5,000IU/kg, 20,000 IU/kg, and 40,000 IU/kg dosing regimens.

[0127]FIG. 80: PD parameters in humans.

[0128]FIG. 81: PK/PD model for rHuEPO in humans.

DETAILED DESCRIPTION OF THE INVENTION

[0129] It is understood that the present invention is not limited to theparticular methodology, protocols, and reagents, etc., described herein,as these may vary. It is also to be understood that the terminology usedherein is used for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention.It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

[0130] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Preferred methods,devices, and materials are described, although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All references citedherein are incorporated by reference herein in their entirety.DEFINITIONS a.m. Ante meridiem, morning AUC Area under the concentrationvs. time curve AUC₍₀₋₁₆₈₎ AUC of epoetin alfa concentration in serumfrom time 0 to 168 hour AUC(RETI) AUC of change in % reticulocytes frombaseline AUC(HEMO) AUC of change in hemoglobin from baseline AUC(RBC)AUC of change in total red blood cell counts from baseline BUN BloodUrea Nitrogen °C. Degrees centigrade C_(max) Maximum concentrationC_(min) Predose trough concentration AUC from time 0 to 168 hour CL/FClearance/bioavailability cm centimeter CRF Case Report Form CV %Coefficient of variation, relative standard deviation d Day D Dose(amount) dL Deciliter g Gram h Hour Hb Hemoglobin Hct Hematocrit HIVHuman Immunodeficiency Virus IRB Institutional Review Board I Negativefeedback mechanism IV Intravenous kg Kilogram L Liter LDH Lactatedehydrogenase LOQ Limit of Quantitation ug Microgram (also μg) mgMilligram min Minute mL Milliliter N/A Not Applicable/Available ngNanogram nm Nanometer NMR Nuclear Magnetic Resonance No. Number NS Notstatistically significant OTC Over-the-counter q.w. Once per week QCQuality control r Correlation coefficient r² Coefficient ofdetermination RBC Red blood cell REF Reference RETI Reticulocyte RIARadioimmunoassay rHuEPO Recombinant human erythropoietin RWJPRI TheRobert Wood Johnson Pharmaceutical Research Institute SC Subcutaneous SDStandard deviation SE Standard error SEM Standard Error of the Mean SGOTSerum glutamic-oxaloacetic transaminase (AST) SGPT Serumglutamic-pyruvic transaminase (ALT) t_(max) Time of maximumconcentration t.i.w. Three-times-a-week TIBC Total iron binding capacityt_(1/2) Elimination half-life WBC White Blood Cell WHOART World HealthOrganization Adverse Reaction Terminology

[0131] EPO, as defined herein, refers to any molecule that specificallystimulates terminal differentiation of red blood cells fromhematopoietic stem cells and stimulates the production of hemoglobin.For example, but not to limit the scope of the present invention, EPOmolecules may include small organic or inorganic molecules, synthetic ornatural amino acid peptides, purified protein from recombinant ornatural expression systems, or synthetic or natural nucleic acidsequences, or any chemical derivatives of the aforementioned. Specificexamples of erythropoietin include, Epoetin alfa (EPREX®, ERYPO®), Novelerythropoiesis stimulating protein (NESP) (a hyperglycosylated analog ofrecombinant human erythropoietin (Epoetin) described in European patentapplication EP640619), human erythropoietin analog—human serum albuminfusion proteins described in the international patent applicationWO9966054, erythropoietin mutants described in the international patentapplication WO9938890, erythropoietin omega, which may be produced froman Apa I restriction fragment of the human erythropoietin gene describedin U.S. Pat. No. 5,688,679, altered glycosylated human erythropoietindescribed in the international patent application WO9911781, PEGconjugated erythropoietin analogs described in WO9805363 or U.S. Pat.No. 5,643,575. Specific examples of cell lines modified for expressionof endogenous human erythropoietin are described in international patentapplications WO9905268 and WO9412650. A preferred form of EPO ispurified, recombinant EPO. For example, purified, recombinant EPO isdistributed under the trademarks of EPREX®, PROLEASE®, or ERYPO®.Specifically. Epoetin alfa (EPREX®, ERYPO®) is a sterile clear,colorless, aqueous solution for injection, that is provided inprefilled, single-use syringes containing either 4,000 or 10,000 IUepoetin alfa (a recombinant human erythropoietin) and 2.5 mg/ml humanserum albumin in 0.4 ml (4,000 IU syringe) or 1.0 ml (10,000 IU syringe)of phosphate buffer.

[0132] EPO also includes those proteins that have the biologicalactivity of human erythropoietin, as well as erythropoietin analogs,erythropoietin isoforms, erythropoietin mimetics, erythropoietinfragments, hybrid erythropoietin proteins, erythropoietin receptoragonists, renal erythropoietin, oligomers and multimers of the above,homologues of the above, and muteins of the above, regardless of thebiological activity of the same, and further regardless of the method orsynthesis or manufacture thereof including but not limited to naturallyoccurring, recombinant, synthetic, transgenic, and gene activatedmethods, e.g., EPO-like proteins. Such EPO and methodologies forproduction are described in, e.g., U.S. Pat. Nos. 5,716,644, 5,674,534,5,916,597, 6,048,524, 5,994,127, 5,955,422, 5,856,298, 5,756,349,5,621,080, 5,547,933, and 5,441,868.

[0133] SC EPO administration, as defined herein, refers to the deliveryof a desired dosage of EPO via a medication delivery device. Themedication delivery device can penetrate the epidermis of the individualto be treated, and results in introducing the desired dosage of EPO intothe tissues of the individual. The delivery device of the presentinvention may include, but is not limited to any traditional hypodermicneedle injectors, air-powered needle-less injectors, jet injectors, orgas-pressured needle-less injectors (see, e.g., U.S. Pat. Nos.5,730,723, 5,891,086, 5,957,886, and 5,851,198.)

[0134] Threshold level, as defined herein, refers to a serum EPOconcentration at which serum EPO concentrations sustained above thislevel will promote the differentiation of young RBCs into mature RBCs.Conversely, serum EPO concentrations maintained below threshold levelwill not lead to the maturity of reticulocytes into RBCs.

[0135] A patient, as described herein, includes individuals who requiredue to a disease state, treatment regimen, or desire, an increase inhematocrit, RBC number, or oxygen capacity.

[0136] Infrastructure domain, as described herein, includes theoperational aspects of the system which are largely transparent to theuser of the system. Examples of infrastructure domain items includeservices which implement concurrency, transaction support, datastructure support, and security services, etc.

[0137] Business domain, as described herein, includes all of theoperations and logic concerning the actual functionality of the systemas it regards the actual business models and methods being implementedby the system. For example, if the business model was providing an EPOdosing regimen and selling the dosed EPO, the business domain logicwould include all of the aspects of running the business. This wouldinclude manufacturing, buying and selling EPO, checking and establishingcredit for customers, etc. It is important to note that the businessdomain does not include support for infrastructure-related activities.

[0138] A preferred embodiment of the present invention describes adosing regimen wherein EPO is administered about 40,000 IU/kg once aweek for two consecutive weeks. The first dose of EPO facilitates theproduction of reticulocytes from RBC progenitor cells. The second doseof EPO is administered to coincide with the reticulocyte pharmacodynamicprofile of the patient. The second dose of EPO will be administered 6-10days following the initial dose, and preferably at the time when thereticulocyte concentration peaks following the first EPO dose.

[0139] This aspect of the present invention relates to the stimulationof the proliferation of erythroid progenitor cells by EPO and therelease of reticulocytes in the blood circulation. After single doseadministration, reticulocytes peaked at times ranging from 6 to 12 daysand returned to predose levels at times up to 15 days. The lifespan ofcells in reticulocyte stage is about 1 to 2 days in the bloodcirculation. Therefore, one skilled in the art would expect young redcells to appear in the blood circulation in about 7 to 14 days afterdosing. It is hypothesized that EPO is required for the maturation ofthe young red cells into mature red cells during Days 7 to 14 after theinitiation of the EPO therapy. Mature red cells have average life spanof 120 days in healthy subjects. The life span of mature red cells couldbe shorter in patients of chronic anemia or other disease states.

[0140] Another preferred embodiment of the present invention comprises adosing regimen wherein EPO is administered in a dosing cycle comprisingtwo or more cycles, in which EPO is administered once a week for twoconsecutive weeks. The length of time between dosing cycles preferablycoincides with the lifespan of RBCs. The lifespan of RBCs is typically120 days, however this may time may vary due to a disease state ortreatment regimen. Therefore, preferably, the subsequent cycle of EPOwill preferably be administered 60-120 days following the previous dose.

[0141] A preferred embodiment of the present invention describes apharmacokinetic model for serum EPO concentrations in healthy volunteersfollowing intravenous (IV) and subcutaneous (SC) dosing as well as apharmacodynamic model of SC EPO causing changes in reticulocyte, RBCnumbers and Hb concentrations in blood. In addition, specific exampleswill follow utilizing the PK/PD models of the present invention. Theseinclude: determining expected differences in hemoglobin responses forvarious dosage regimens of rHuEPO; assessing the effects of subject bodyweight on the expected response to maintenance therapy with rHuEPO; andinvestigating whether cancer patients respond to the same extent ashealthy volunteers to rHuEPO treatment.

[0142] Pharmacokinetic Model and Analysis

[0143] Data describing modeling and SC rHuEPO pharmacokinetics wereobtained from clinical studies performed by The Robert Wood JohnsonPharmaceutical Research Institute (RWJPRI). This consisted of twocomparable, open-label, randomized, parallel, placebo-controlled studiesin healthy volunteers where rHuEPO was administered as eight single SCdoses of EPREX®: 300, 450, 600, 900, 1200, 1350, 1800, 2400 IU/kg and asmultiple dosage regimens: 150 IU/kg three times a week for four weeksand 600 IU/kg one per week for four weeks. Each treatment group had 5subjects.

[0144] The measured rHuEPO concentrations after rHuEPO administrationwere corrected for baseline values because the radioimmunoassay usedcould not distinguish between endogenous EPO and rHuEPO. The baselineEPO concentration for each subject was determined by averaging thepredose values (10, 20 and 30 min). This value was subtracted from thepost-dose values at each time point to obtain the corrected serum rHuEPOconcentration. The mean of the corrected concentrations for all subjectswas used for data analysis. Any measurement below the limit of assaydetection (7.8 IU/I) was not used as a data point. The intravenous datawere also corrected for baseline EPO levels.

[0145] From preliminary analysis of IV data obtained from theliterature, a one-compartment model was found to be adequate. Thedisposition of rHuEPO has been reported to be nonlinear mainly becauseof a dose-dependant decrease in clearance (see, e.g., Macdougall et al.,1991. Clin. Pharmacokinet. 20:99-113.). Therefore, the Michaelis-Mentenfunction was used to describe rHuEPO disposition. The IV data for rHuEPOconcentrations (C_(EPO)=Ap/Vd) versus time were fitted with thefollowing equation: $\begin{matrix}{\frac{{Ap}}{t} = {{- \left( \frac{V\quad \max}{{{Km}*{Vd}} + {Ap}} \right)}*{Ap}}} & (1)\end{matrix}$

[0146] where Ap is the amount of rHuEPO in the body, Vmax is thecapacity of the process, Km is the affinity constant or the plasmarHuEPO concentration at which the elimination rate reaches one-halfVmax, and Vd is the volume of distribution.

[0147] The IV concentration-time profiles for the various doses areshown in FIG. 2. A one-compartment model with non-linear disposition wasused to describe the kinetics of rHuEPO. Notably, a two-compartmentmodel might better fit the IV data at early times; nonetheless, it wouldproduce greater complexity in overall data fitting. Consequently, aone-compartment model was chosen as it gave acceptable fittings andserved the purpose. Also, since rHuEPO is a 30 KDa protein, it can beexpected to be restricted to the intravascular compartment, thusjustifying the choice of a one-compartment model.

[0148] The parameters obtained by the fittings are listed in FIG. 3. TheVd (0.0558 L/kg) and Vmax/Km (i.e., CL at low doses: 0.0066 L/hr/kg)obtained are in the range of reported literature estimates (see e.g.,Macdougall et al., supra and Lappin et al., 1996. Clin. Lab Haem.18:137-1458.) The large Km value indicates that EPO disposition is onlymildly non-linear and dose-dependant elimination would become importantonly at high doses. Studies in rats have suggested that binding ofrHuEPO to receptors in bone marrow and spleen contribute to thesaturable elimination of rHuEPO (see, e.g., Kato et al., 1997, J.Pharmacol. Exp. Ther. 283:520-27.).

[0149] The pharmacokinetics of SC EPO was found to be best characterizedby a dual-absorption model with a fast zero-order input of most of thedrug (87% of that absorbed) followed by slow first-order absorption of asmall part of the drug (13% of that absorbed). The bioavailability wasfound to increase with dose (ranging 46 to 100%) contributingsubstantially to the nonlinearity in the kinetics. The differentialequations for the model (FIG. 4) are shown as follows: $\begin{matrix}{{\frac{{Ap}}{t} = {{{ko}\left( {0 - \tau} \right)} + {k_{1}\left( {t > \tau} \right)} - {\left( \frac{V\quad \max}{{{Km}*{Vd}} + {Ap}} \right)*{Ap}}}}{where}{{ko} = {{0\quad {when}\quad t} > \tau}}{{ko} = {{\frac{F*\left( {1 - {Fr}} \right)*{Dose}}{\tau}\quad {when}\quad 0} < t \leq \tau}}{k_{1} = {{0\quad {when}\quad t} \leq \tau}}{and}{k_{1} = {{{ka}*F*{Fr}*{Dose}*^{{- {({{ka}*{({t - \tau})}})}}\quad}\quad {when}\quad t} > \tau}}} & (2)\end{matrix}$

[0150] The rHuEPO was assumed to be 100% bioavailable on IVadministration. The bioavailability after SC dosing is represented bythe parameter F. Accordingly, the amount of rHuEPO associated with thefirst-order process is given by F*Fr*Dose while that absorbed by thezero-order process is given by F*(1−Fr)*Dose.

[0151] All fittings were done using the ADAPT II software (see, e.g.,Argenio et al., 1998. ADAPT II User's Guide, Biomedical SimulationsResource, University of Southern California, Los Angeles), although anyappropriate software also could have been employed. Estimation ofparameters was done using least-squares fitting by the MaximumLikelihood method and the extended least-squares variance model used isgiven by:

V(l)=Inter ² *Y(1)sigma+0.0001 where Y(1)=Ap/Vd

[0152] Simultaneous fitting of the eight single SC doses and five IVdose levels was performed. During initial fittings, Vmax, Km, Vd, ka, Frand variance parameters were kept constant across doses while τ and Fwere allowed to vary with dose. Results indicated that τ could be fixedas a single value up to the 1350 IU/kg dose whereas for the last twodoses, a higher τ value was optimal. Bioavailability F was found toincrease proportionately with dose in the range of doses tested and wasdescribed by a linear equation (r²=0.9713) as follows:

F=0.3884+0.00024952*Dose  (3)

[0153] For linear regression, the F value for the 450 IU/kg dose wasexcluded as it appeared to be an outlier. Final fittings were done usingthis function to set F values across doses and τ was fixed to be 44hours for all doses except the last two for which T was set as 60 hours.This made it possible to describe all the data using a single set ofparameters for Vmax, Km, Vd, ka, Fr, and the variance parameters. The600 IU/kg/week multiple dosing data were simulated using the sameparameters with F set to 60% as it gave optimal fittings. For the 150IU/kg t.i.w. regimen, F was fixed at 25%. In addition, for this lowdose, it was necessary to increase the Vd (0.1193 l/kg) and set the τ as18 hrs.

[0154]FIGS. 5A and 5B show the mean rHuEPO concentration-time profilesfor the different single SC doses. Visual inspection of the SC dataclearly indicates flip-flop kinetics since the terminal slope is muchflatter compared to the IV monoexponential decline. Hence, a first-orderabsorption rate constant was assigned to capture the terminal phase. Thedata also shows that rHuEPO concentrations rapidly reach the peak Cmaxwithin one day, thereby indicating that there must be a fasterabsorption process as well. This rapid upcurve was accounted for by azero-order input process. The terminal slopes across all of the doseswere found to be parallel indicating that a single first-orderabsorption rate ka could account for this phase for all doses. Thefraction of dose associated with the slow first-order absorption processwas only 13.1%. Thus, the majority of the bioavailable dose is rapidlyabsorbed within 2-3 days by the zero-order component. The first-orderabsorption rate ka accounts for a slow continuous release of rHuEPO fromthe subcutaneous site. A similar dual-absorption rate model was used tocharacterize the absorption kinetics of another macromolecule, IL-10,following SC dosing (see, e.g., Radwanski et al., 1998. Pharm. Res.15:1895-1902.)

[0155]FIG. 6 provides the plot of AUC versus dose for the differentsingle SC doses. The greater than proportional increase in AUC withincreasing dose indicates that either CL or bioavailability or both arechanging with dose. Elimination of rHuEPO was found to be only verymildly nonlinear. On the other hand, F was found to increase linearlywith dose (FIG. 7) and turned out to be the main factor responsible forthe disproportionate increase in AUC with SC dose. At the last twohighest doses, reduced CL was also contributing to non-linearity. FIG. 8lists the F values for all the doses obtained by deconvolution and byfitting the data from individual doses to the proposed model. Theestimates obtained using both methods are very similar, thus indicatingthat the pharmacokinetic model of the present invention can adequatelyaccount for the nonlinearity due to changing values of F.

[0156] The cause of the incomplete and nonlinear bioavailability of SCrHuEPO is not known. For example, the protein IL-10 exhibits only 42%bioavailability on SC dosing with loss assumed to be due to the effectsof proteolytic enzymes (see, e.g., Id.). In turn, these enzymes may besaturable at higher concentrations of peptide or protein substrates. Thedual absorption process may be due to the role of the lymphatics incontrolling access of macromolecules after SC dosing. The rapid earlyabsorption phase may be caused by leakage of a major part of the doseinto local blood vessels while the later phase may be related to slowentry via the lymphatic system.

[0157] Simulations of rHuEPO concentrations versus time for the 150IU/kg t.i.w. and 600 IU/kg/week multiple-dosing regimens are shown inFIG. 9. The pharmacokinetic model of the present invention, with most ofthe parameters fixed to the value obtained from fittings of the singledoses, accurately describes the multiple dosing data. For the lowermultiple-dosing regimen of 150 IU/kg, the Vd had to be increased. Thismay be due to non-linearities involved in distribution when thebioavailable amount is so low. The same change in Vd may also facilitatea better fitting of the 10 IU/kg IV dose where the maximumconcentrations are in the same range.

[0158] Pharmacodynamic Model and Analysis

[0159] Data describing the pharmacodynamics of reticulocytes, RBCnumbers, and Hb levels in blood were obtained from clinical studiesperformed by RWJPRI. This consisted of two comparable, open-label,randomized, parallel, placebo-controlled studies in healthy volunteerswhere rHuEPO was administered at eight single SC doses of EPREX®: 300,450, 600, 900, 1200, 1350, 1800, 2400 IU/kg and as multiple dosageregimens: 150 IU/kg three times a week for four weeks and 600 IU/kg oneper week for four weeks. Each treatment group had 5 subjects. Thepharmacodynamic end points measured were the number of reticulocytes,RBCs in blood, and hemoglobin count.

[0160] The pharmacodynamic data were described using a cell productionand loss model, which describes the changes in cell numbers with timeafter rHuEPO administration. According to this model, all cells involvedin the process of erythropoiesis are assumed to be produced in a zeroorder fashion (k0): they live for a fixed time period at the end ofwhich they die or are converted to other cells. As a result, the cellsare lost at a rate which is exactly equal to the rate at which they wereborn, except that their elimination is delayed by a time period which isequal to the life-span of the cell. It is assumed that the lifespan ofany single set of cells is constant with respect to time and is the samefor each cell of that type.

[0161]FIG. 10 provides a schematic representation of a PD model of thepresent invention. The life spans of each precursor cell (TP),reticulocyte (R_(L)), and red blood cell (RBC_(L)) are indicated. Thecompartments reflect the pools of erythroid progenitor cells (P),reticulocytes (R), red blood cells (RBC) and hemoglobin (Hb) in theblood. Stimulation of erythropoiesis by the administered rHuEPO (Cp(t))is given by the Hill function (S(t)) acting on the production ofprecursor cells in the marrow.

[0162] The differential equations for the model are as follows:

dP/dt=k0[S(t)−S(t−TP)]  (4)

dR/dt=k0[S(t−TP)−S(t−TP−R _(L))]  (5)

dRBC/dt=k0[S(t−TP−R _(L))−S(t−TP−R _(L) −RBC _(L))]  (6)

[0163] where $\begin{matrix}{{S(t)} = \frac{S_{\max}*\left( {{{Cp}(t)} + C_{bs}} \right)}{{SC}_{50} + {{Cp}(t)} + C_{bs}}} & (7) \\{{S\left( {t - {TP}} \right)} = \frac{S_{\max}*\left( {{{Cp}\left( {t - {TP}} \right)} + C_{bs}} \right)}{{SC}_{50} + {{Cp}\left( {t - {TP}} \right)} + C_{bs}}} & (8) \\{{S\left( {t - {TP} - R_{L}} \right)} = \frac{S_{\max}*\left( {{{Cp}\left( {t - {TP} - R_{L}} \right)} + C_{bs}} \right)}{{SC}_{50} + {{Cp}\left( {t - {TP} - R_{L}} \right)} + C_{bs}}} & (9) \\{{S\left( {t - {TP} - R_{L} - {RBC}_{L}} \right)} = \frac{\begin{matrix}{S_{\max}*} \\\left( {{{Cp}\left( {t - {TP} - R_{L} - {RBC}_{L}} \right)} + C_{bs}} \right)\end{matrix}}{\begin{matrix}{{SC}_{50} +} \\{{{Cp}\left( {t - {TP} - R_{L} - {RBC}_{L}} \right)} + C_{bs}}\end{matrix}}} & (10)\end{matrix}$

[0164] S_(max) is the maximum possible stimulation of reticulocyteproduction by rHuEPO and SC₅₀ is the plasma concentration of rHuEPOwhich can cause one-half maximum stimulation. The parameter Ks isdefined as Ks=k0*S_(max) and it signifies the maximum possibleproduction rate of cells upon stimulation by rHuEPO.

[0165] The SC₅₀ was also used as a threshold rHuEPO concentration forformation of RBC from reticulocytes. It was assumed that when rHuEPOconcentrations fall below this limit, the reticulocytes are notconverted to erythrocytes. For this process, the stimulation functionfor model equation (6) was adjusted as follows:

If Cp(t)≦SC ₅₀, then Cp(t−TP−R _(L)=0)  (11)

and Cp(t−RBC _(L))≦SC ₅₀, then Cp(t−TP−R _(L) −RBC _(L))=0  (12)

[0166] The baseline conditions (steady-state levels) are defined asfollows: $\begin{matrix}{{\frac{{Pss}}{t} = 0},{\frac{{Rss}}{t} = {{0\quad {and}\quad \frac{{RBCss}}{t}} = 0}}} & (13)\end{matrix}$

[0167] As a result, the initial condition itself defines thesteady-state levels.

Pss=P(0), Rss=R(0) and RBCs=RBC(0)  (14)

[0168] The precursor steady-state conditions can be defined in explicitform as follows:

Pss=kin*TP  (15)

[0169] Since the baseline condition for the precursor compartment isunknown, a value of 14*10¹⁰ cells/L/hr was assigned using the aboveequation and literature estimates of K_(in)(19).

[0170] The change in hemoglobin levels was modeled by simply using aproportionality factor Hb_(cell), which represents the hemoglobincontent per cell (reticulocyte or RBC).

Hb(t)=Hb _(cell) *No.cells(t)  (16)

[0171] As depicted in FIG. 1, erythropoiesis involves a cascade ofevents. The precursor compartment in the model is representative of allcells in the bone marrow involved in this process which are eventuallyconverted to reticulocytes. The time TP thus serves as an average lengthof time taken for the earliest precursor cell stimulated by rHuEPO toundergo the cascade of differentiation processes to finally getconverted to a reticulocyte. In other words, it accounts for the timedelay seen for reticulocytosis to be initiated by rHuEPO. Once areticulocyte is formed, it exists for a time equal to R_(L) at whichpoint it is converted to a RBC. It is assumed that the primary way bywhich a reticulocyte could be lost is by conversion to an erythrocyte,except for the subthreshold condition. The model does not account forrandom destruction of cells such as bleeding. Hence, the production andelimination rate of all these cells can be represented by a singlezero-order rate constant k0. Once an RBC is produced, it in turnsurvives for a period of RBC_(L) days after which it disappears fromblood.

[0172] The transformation of a reticulocyte to RBC occurs partly in thebone marrow and partly in the blood over a period of 72 hours (see,e.g., Jusko, 1994. Clin. Pharmacol. Ther. 56:406-19.) Reticulocytes areformed in the marrow and by diapedesis, they pass into the circulationwhere these immature cells develop for a period of 24-48 hr before beingtransformed to erythrocytes (see, e.g., Guyton, 1996. Textbook ofMedical Physiology, W. B. Saunders, Philadelphia.) Since rHuEPO is knownto stimulate the premature release of reticulocytes from the marrow, itwas assumed that these newly born cells spend their whole life span of72 hours in the blood under rHuEPO stimulation. Consequently, the lifespan of reticulocyte, R_(L) was fixed to be 72 hours. The RBC life span,RBC_(L) was fixed to be 120 days and the Hb content per cell was fixedto be 29.5 pg/cell based on literature values (see, e.g., Jusko, supraand Guyton, supra.) Both reticulocytes and RBC are assumed to contributeto the overall Hb content of blood.

[0173] Furthermore, the new cell production (rate Ks) and loss model wasused to obtain estimates of the SC₅₀ and Ks. Reticulocytes and red bloodcells were assumed to have 3 day (72 hour) and 120 day (2880 hour)life-spans. The time lag in appearance of reticulocytes in blood wasestimated by introduction of a precursor compartment representing theprogenitor cells. A Ks value of 0.3709×1010 cells/L/hr was obtainedyielding roughly an S_(max) of 2 which reflects a moderate maximumstimulation of erythropoiesis. The SC₅₀ value obtained was 23 IU/Lindicating that low serum rHuEPO concentrations were sufficient tomaintain stimulation. A threshold concentration assumed equal to SC₅₀ ofrHuEPO was found to be essential for conversion of reticulocytes toerythrocytes in the blood. Single doses of rHuEPO up to 900 IU/kg wereincapable of maintaining rHuEPO levels above this threshold during thetime of RBC production. This explains the lack of increase in RBCnumbers in spite of stimulation in reticulocyte production afteradministration of the four lower single SC doses, consequently yieldingonly a very slight change in Hb for a brief period of time. Also, thethreshold concept explains the better responsiveness of Hb levels torHuEPO multiple-dosage regimens, which caused a significant improvementin Hb levels. The parallel nature of the study produced variability inresponses to different rHuEPO doses, but a single set of parametersprovided reasonable characterization of responses to the range of dosesand regimens.

[0174]FIG. 11 shows the mean reticulocyte number versus time profilesfor all single SC doses. The reticulocyte counts slightly increasecompared to predose levels immediately at the first sampling point. Thislevel is maintained for 3-4 days after which they steadily start risingtill the peak is reached around 200-300 hour. Then, counts startdeclining rapidly with an apparent half-life of 25 hours to reachbaseline levels by day 22 (528 hours).

[0175] The data and model fittings are shown in FIGS. 12A and 12B.Parameter estimates obtained by fitting the pharmacodynamic equations tothe data are presented in FIG. 13. A Ks of 0.3709×10¹⁰ cells/L/hr wasobtained which translates to 4.451×10¹⁰ cells/day assuming a bloodvolume of 5 L. It is known that 1% of all RBC are destroyed daily andreplaced by reticulocytes in healthy humans yielding an erythrocyteproduction rate (k0) of 2-3×10¹¹ cells/day. Therefore, the estimatedS_(max) is 1.5-2.2 which indicates that rHuEPO can produce a maximum 2.5to 3.2-fold increase in the zero-order production rate of reticulocytes,a relatively modest degree of stimulation which accounts for the slowand limited rise in blood.

[0176] The SC₅₀ of 22.58 IU/L obtained reflects the serum rHuEPOconcentration needed to cause half-maximal stimulation. As long asrHuEPO serum concentrations are maintained above this value, the cellcounts should remain above baseline. Normal erythrocytic progenitorcells, regardless of origin, express less than 1000 EPO-receptors on thecell surface. Binding of EPO to this receptor causes signal transductionevents which ultimately lead to stimulation of the differentiation andproliferation of erythrocytic progenitors in the bone marrow (see, e.g.,Lappin, supra.) In addition, EPO accelerates the release ofreticulocytes from the marrow leading to increased serum reticulocyteand erythrocyte numbers (Id.). The slight increase in levels ofreticulocytes seen after the zero time point may be caused due to theearly release of immature reticulocytes from the marrow which is notaccounted for by the model at these early times.

[0177] The low SC₅₀ value of 22.58 IU/L obtained reflects the fact thatthere are low numbers of receptor sites on erythropoietic cells whichmay be readily saturated so that high doses with rapid delivery may leadto considerable wastage of the bioavailable rHuEPO. An increase in doseor slower delivery facilitates rHuEPO levels being maintained above theSC₅₀ for a longer time and so there is an increase in the extent andduration of stimulation of reticulocyte production. This conceptexplains the results of two recent clinical studies that show that SCrHuEPO is more effective than IV dosing for stimulating production oferythrocytes. In spite of lower bioavailability, the SC doses withprolonged absorption results in more efficient stimulation of RBCproduction (see, Kaufmann, et al., 1998. N. Engl. J. Med. 339:578-83 andBesarab, et al., 1992. Am. Soc. Nephrol. 2:1405-12.)

[0178]FIGS. 12A and 12B show that the pharmacodynamic data for some doselevels are quite variable, which is reflected by the inconsistencies inthe extent of stimulation with increasing dose. For instance, the 600and 1200 IU/kg doses produce slightly higher numbers of cells comparedto the 900 and 1350 IU/kg doses. The pattern of return, however, tobaseline seems to be similar across doses. In any case, the models ofthe present invention capture the trend of responses, considering thevariable nature of the data and the fact that one single set ofparameters could adequately describe the pharmacodynamic data from alldoses. It would be possible to obtain better fittings of the data fromeach dose level by allowing Ks and SC₅₀ to vary for each group. Thiswould be reasonable since these were parallel dose groups, but then theparameters would have to be averaged for purposes of generalization.

[0179]FIG. 14 shows the Hb response versus time after the 8 single SCdoses and the simulations. There is very little, if any, change in theHb levels and this could be explained by the fact that rHuEPOconcentrations fall close to the threshold limit by 7 days, thuspreventing the conversion of newly formed reticulocytes to RBC. Theslight elevation in Hb levels are therefore solely caused by increase inreticulocyte numbers for doses up to 900 IU/kg, after which there aremodest increases in RBC also contributing to the consistently higher Hblevels. This threshold concept might explain the dose-dependant increasein reticulocyte response without significant increases in hematocrit orhemoglobin responses after single intravenous doses up to 1000 IU/kg inhealthy volunteers as reported by Flaharty et al., supra.

[0180]FIGS. 15 and 16 show the model output for the change inreticulocyte, RBC and Hb counts after multiple SC dosing of rHuEPO. Thepharmacodynamic responses for both the dosage regimens are captured. TherHuEPO concentrations are maintained above the threshold for most of theperiod of time, giving the reticulocytes a chance to get converted toRBC, which is reflected as a steady increase in Hb levels aftermultiple-dosing in contrast to the single doses. The fittings of thedifferent single SC doses and simulations of the multiple SC doses showthat maintaining the rHuEPO concentrations above the SC₅₀ byadministration of several smaller doses tends to enable a continuousincrease in Tib levels as opposed to giving the same total dose as asingle shot. In the latter case, the concentrations fall below the SC₅₀much sooner causing a diminished reticulocyte response and moreimportantly, an ineffective Hb response.

[0181] In conclusion, the PK/PD models of the present inventiondemonstrate the importance of dose, dosage regimen, and route ofadministration in controlling rHuEPO responses and can be used as avaluable tool for designing optimal rHuEPO doses and time ofreadministration for various conditions. In addition, the presentinvention provides a computer-based system to tailor the dosage andschedule of the EPO treatment such that the patient receives optimumbenefit in terms of, for example, increased hemoglobin and reticulocyteproduction, or to provide a dosage regimen, for example, once-weekly oronce every two weeks, that suits the patient's needs.

[0182] Business Method

[0183] In a particular embodiment of the present invention, a businessmethod relating to providing a dosing regimen of EPO and sale of thedosed EPO may be implemented. In a specific embodiment, that method maybe implemented through the computer systems of the present invention.For example, a user (e.g., a health practitioner such as a physician ora pharmacist) may access the computer systems of the present inventionvia a computer terminal and through the Internet or other means. Theconnection between the user and the computer system is preferablysecure.

[0184] In practice, the user may input, for example, informationrelating to a patient such as the patient's disease state, hematocrit,hemoglobin concentration, and other factors relating to the patient'sreticulocyte and/or RBC count, such as the desired or optimalreticulocyte or RBC count. The computer system may then, through the useof the resident computer programs, provide one or more appropriate EPOdosing regimens for the patient. The computer program, via the userinterface, may also provide pricing and cost comparisons for differentEPO or EPO-like drugs, in conjunction with, or separate from,appropriate dosing regimens for those EPO or EPO-like drugs.

[0185] A computer system in accordance with a preferred embodiment ofthe present invention may be, for example, an enhanced IBM AS/400mid-range computer system. However, those skilled in the art willappreciate that the methods and apparatus of the present invention applyequally to any computer system, regardless of whether the computersystem is a complicated multi-user computing apparatus or a single userdevice such as a personal computer or workstation. Computer systemsuitably comprises a processor, main memory, a memory controller, anauxiliary storage interface, and a terminal interface, all of which areinterconnected via a system bus. Note that various modifications,additions, or deletions may be made to the computer system within thescope of the present invention such as the addition of cache memory orother peripheral devices.

[0186] The processor performs computation and control functions of thecomputer system, and comprises a suitable central processing unit (CPU).The processor may comprise a single integrated circuit, such as amicroprocessor, or may comprise any suitable number of integratedcircuit devices and/or circuit boards working in cooperation toaccomplish the functions of a processor. The processor suitably executesthe PK/PD modeling computer programs of the present invention within itsmain memory.

[0187] In a preferred embodiment, the auxiliary storage interface allowsthe computer system to store and retrieve information from auxiliarystorage devices, such as magnetic disk (e.g., hard disks or floppydiskettes) or optical storage devices (e.g., CD-ROM). One suitablestorage device is a direct access storage device (DASD). A DASD may be afloppy disk drive which may read programs and data from a floppy disk.It is important to note that while the present invention has been (andwill continue to be) described in the context of a fully functionalcomputer system, those skilled in the art will appreciate that themechanisms of the present invention are capable of being distributed asa program product in a variety of forms, and that the present inventionapplies equally regardless of the particular type of signal bearingmedia to actually carry out the distribution. Examples of signal bearingmedia include: recordable type media such as floppy disks and CD ROMS,and transmission type media such as digital and analog communicationlinks, including wireless communication links.

[0188] The computer systems of the present invention may also comprise amemory controller, through use of a separate processor, which isresponsible for moving requested information from the main memory and/orthrough the auxiliary storage interface to the main processor. While forthe purposes of explanation, the memory controller is described as aseparate entity, those skilled in the art understand that, in practice,portions of the function provided by the memory controller may actuallyreside in the circuitry associated with the main processor, main memory,and/or the auxiliary storage interface.

[0189] Furthermore, the computer systems of the present invention maycomprise a terminal interface that allows system administrators andcomputer programmers to communicate with the computer system, normallythrough programmable workstations. It should be understood that thepresent invention applies equally to computer systems having multipleprocessors and multiple system buses. Similarly, although the system busof the preferred embodiment is a typical hardwired, multidrop bus, anyconnection means that supports bidirectional communication in acomputer-related environment could be used.

[0190] The main memory of the computer systems of the present inventionsuitably contains one or more computer programs relating to the PK/PDmodeling of EPO administration and an operating system. Computer programin memory is used in its broadest sense, and includes any and all formsof computer programs, including source code, intermediate code, machinecode, and any other representation of a computer program. The term“memory” as used herein refers to any storage location in the virtualmemory space of the system. It should be understood that portions of thecomputer program and operating system may be loaded into an instructioncache for the main processor to execute, while other files may well bestored on magnetic or optical disk storage devices. In addition, it isto be understood that the main memory may comprise disparate memorylocations.

[0191] Other objectives, features and advantages of the presentinvention will become apparent from the following specific examples. Thespecific examples, while indicating specific embodiments of theinvention, are provided by way of illustration only. Accordingly, thepresent invention also includes those various changes and modificationswithin the spirit and scope of the invention that may become apparent tothose skilled in the art from this detailed description.

Example 1 Human Pharmacokinetics and Bioavailability for EPO

[0192] The following example of the present invention provides a summaryof the PK/PD data that support a 40,000-IU q.w. dosing regimen. The dataare derived from both the literature and four clinical studies conductedby RWJPRI, Raritan, N.J. Three studies were conducted underInvestigational New Drug BB-IND-2318, and one study was conducted in theUK. A brief overview of the studies is given in FIG. 17.

[0193] The clinical pharmacokinetic studies included in this technicalsummary are described FIG. 18A-18D, and the pharmacokinetic data fromthese studies are summarized in FIG. 19. The analytical methods used forthe determination of EPO concentration in serum are summarized, infra,and FIG. 20.

[0194] Clinical Study EPO-PHI-373 (FIG. 17) provides the data to supportthe 40,000 IU once weekly dosing regimen. Clinical Study EPO-PHI-370(FIG. 17), which has a similar design as Clinical Study EPO-PHI-373.

[0195] In Clinical Study EPO-PHI-370, there were randomization issues,fluctuating hemoglobin levels, and many subjects were iron-deficient atinitial. Error in the randomization at the study site resulted in anunequal distribution of males and females in each treatment group, andcontributed to an imbalance in mean baseline hemoglobin values. A reviewof hematology values revealed a substantial fluctuation in hemoglobinfor a number of subjects between screening and baseline evaluations.Further investigation revealed laboratory inconsistencies relating toequipment calibration. To appropriately confirm the positive findings ofthis study, it was decided that a repeat study would be conducted.Clinical Study EPO-PHI-373 was the repeat of Clinical Study EPO-PHI-370.

[0196] Clinical Studies EPO-PHI-358 (FIG. 17) and EPO-PHI-359 (FIG. 17)are two pilot studies conducted to investigate the dose-proportionalityof EPO and to obtain preliminary pharmacokinetic and pharmacodynamicinformation after different single and multiple doses.

[0197] General Analytical Methods for In Vivo Studies

[0198] Summary

[0199] Bioanalytical methods were developed and validated to support theclinical program for EPO conducted by RWJPRI, Raritan, N.J. The originalmethod used to determine EPO concentrations in human serum was aradioimmunoassay (RIA) (RWJPRI Study Nos DM92001 and DM96023). Thismethod was successfully transferred to PPD Development (PPD), RichmondVa., the Contract Research Organization which conducted the analysis ofthe supportive clinical study (EPO-PHI-370). This RIA method was fullycross-validated with an enzyme-linked immunosorbent assay (ELISA) at PPDfor the analysis of the pivotal study (EPO-PHI-373). To test thecomparability between the RIA and the ELISA, a set of 16 pooled humansubject samples was prepared by RWJPRI and analyzed at PPD in both theELISA and RIA. The ELISA provided improvements in accessibility ofreagents, time of analysis and extended range of standard curve withoutloss of sensitivity.

[0200] Assay Type

[0201] A radioimmunoassay was used for the quantitation of EPO tosupport Clinical Studies EPO-PHI-358, EPO-PHI-359 (conducted at RWJPRI)and EPO-PHI-370 (conducted at PPD). An ELISA was used for thequantitation of EPO in Clinical Study EPO-PHI-373 (conducted at PPD).

[0202] The RIA method was originally developed by Diagnostic SystemsLaboratories (DSL), Webster, TX, for the quantitative determination ofEPO and the results were compared to those obtained with a RWJPRImethod. The commercially available RIA is a double-antibody, competitivemethod that uses a rabbit polyclonal antiserum to human urinary EPO asthe primary antibody and an ¹²⁵1-labeled human urinary EPO as tracer.The procedure follows the basic principle of adioimmunoassay wherebythere is competition between a radioactive and a nonradioactive antigenfor a fixed number of antibody binding sites. The amount of ¹²⁵I-labeledEPO bound to the antibody is inversely proportional to the concentrationof EPO present in the serum. The separation of free and bound antigen iseasily and rapidly achieved by using an accelerated double antibodypolyethylene glycol system. The major in-house modification of the DSLkit was substitution of recombinant human EPO for urinary EPO instandards and spiked quality control samples. Standard concentrationsused in the assay were 7.8, 15.6, 31.3, 50, 62.5, 100, and 125 mU/mL.This exact method was transferred to and validated by PPD.

[0203] The Immunochemistry Department of PPD validated the ELISA fordetermination of EPO concentrations in human serum. This ELISA is adirect, double-antibody sandwich assay developed by R&D Systems, Inc,Minneapolis, Minn., for the quantitative determination of EPOconcentrations in plasma or serum. Microtiter wells, precoated withmonoclonal (murine) antibody specific for r-HuEPO are used to captureEPO. The bound EPO is labeled with anti-EPO polyclonal (rabbit) antibodyand horseradish peroxidase (conjugate). An optical signal is producedwith the addition of tetramethylbenzidine/buffered hydrogen peroxide(substrate). The amount of color generated is directly proportional tothe concentration of EPO in sample or standard. The major in-housemodification of the R&D kit was use of in-house recombinant human EPO instandards and spiked quality control samples. Standard concentrationsused in the assay were 7.8, 15.6, 31.3, 50, 62.5, 100, 125, and 250mU/mL.

[0204] Range of Standard Curves

[0205] The RIA used for the Clinical Studies EPO-PHI-358, EPO-PHI-359,and EPO-PHI-370 had a standard curve range of 7.8 to 125 mU/mL using a0.1-mL sample aliquot. The precision of the standard curve at RWJPRI was<5.8% and at PPD, <10.8%. The ELISA used to assay clinical samples fromClinical Studies EPO-PHI-373 demonstrated a concentration range of 7.8to 250 mU/mL using a 0.1-mL sample aliquot with a precision of <5.3%.

[0206] Lower Limit of Quantitation

[0207] The lower limit of quantitation (LLOQ), the lowest measurablestandard concentration which could be accurately and preciselyquantified, was 7.8 mU/mL in both the RIA and the ELISA.

[0208] Quality Controls

[0209] Quality control samples (QCs) for both methods were prepared inblank serum to reflect the expected concentrations in the study. QCconcentrations assayed during the validation of the RIA at RWJPRI were35, 60 100, 300, 1000, and 2000 mU/mL. QC concentrations assayed duringthe validation of the RIA at PPD were 100, 500, 2000, and 5000 mU/mL.The QC concentrations for the ELISA were 7.8, 20, 100, 500, 2000, and5000 mU/mL. At least three levels of QCs in human serum were assayedwith study samples during daily sample analysis.

[0210] A set of 16 pooled human serum samples from a previous clinicalstudy was prepared by RWJPRI and the blinded samples were shipped to PPDto be assayed in both the RIA and the ELISA. The relative percentdifference (RPD) calculated between the values obtained by both methodswas determined to be 15.8% (for the lowest level of QC) or better.

[0211] Dilutional linearity was also demonstrated since the expectedconcentrations of some samples were above the highest standard curveconcentration. During the validation of the RIA at RWJPRI linearity upondilution was established using serial 1:30- to 1:256-fold dilutions ofQC samples. At PPD dilutional linearity was demonstrated during RIAvalidation with 1:20- to 1:200-fold dilutions in QC samples and from1:20-to 1:100-fold dilutions in in vivo samples from one subject withhigh EPO concentrations. During the ELISA validation the dilutionallinearity was established using 1:50-, 1:100-, and 1:200-fold dilutionsof the 5000-mU/mL QC.

[0212] Recovery

[0213] The assay was run without sample extraction, therefore recoveryassessment was not required.

[0214] Accuracy and Precision

[0215] In the RIA, the intra-assay accuracy (percent difference betweenmeasured concentration and target concentration) ranged from 102.7 to127% at RWJPRI and 95.5 to 100.3% at PPD. The interassay accuracy rangedfrom 81.7 to 109.8% at RWJPRI and 100 to 105.6% at PPD. In the ELISA theintra-assay accuracy ranged from 87.9 to 109.9% and the interassayaccuracy ranged from 83.6 to 116%.

[0216] In the RIA, the intra-assay precision (percent coefficient ofvariation) was ≦8.2% at RWJPRI and ≦5.3% at PPD. The interassayprecision was ≦15.1% at RWJPRI and ≦7.2% at PPD. In the ELISA, theintra-assay precision was ≦10% and the interassay precision was ≦12.9%.

[0217] Stability

[0218] The stability of EPO in human serum was demonstrated for 20months at −20° C., at room temperature for 2 hours and during fourfreeze/thaw cycles.

[0219] Data Analysis and Acceptability Criteria

[0220] A validated four parameter logistic curve was used to determineunknown serum sample concentrations of EPO from the standard curve inboth assays. The data from the RIA performed at RWJPRI was reduced usingMicromedic software (ICN Biomedicals, Inc., Costa Mesa, Calif.). Thedata from the RIA and the ELISA performed at PPD was reduced using thePPD Development Oracle 7.3 proprietary RICORA database. The RIA assayswere acceptable when the QCs were within 20% of nominal and thedifference between the count per minutes of the replicates was <6%. TheELISA assays were acceptable when the QCs were within 20% for bothaccuracy and precision.

[0221] Summaries of Individual Studies

[0222] Pilot Exploratory Studies, EPO-PHI-358 and EPO-PHI-359

[0223] Clinical Study EPO-PHI-358 and Clinical Study EPO-PHI-359 weretwo pilot exploratory studies designed to investigate thedose-proportionality of EPO after SC administration. Because of thesimilarity in study design, data from the two studies were pooled tomaximize the dose range for pharmacokinetic and pharmacodynamicanalyses.

[0224] The objective of Clinical Study EPO-PHI-358 was to determine thepharmacokinetic and pharmacodynamic profiles of EPO after a single 450-,900-, 1350-, or 1800-IU/kg subcutaneous dose of EPO and to compare theseprofiles to that of the typical 150-IU/kg t.i.w.×4-week dosing regimen.The objective of Clinical Study EPO-PHI-359 was to determine the PK andPD profiles of EPO after a single 300-, 600-, 1200-, or 2400-IU/kg SCdose of EPO and to compare these profiles to that of the 600-IU/kgq.w.×4-week dosing regimen. There was a placebo group in each study.

[0225] The EPO formulation used in these two studies was a 40,000 IU/mL,preservative-free phosphate buffered solution containing 2.5 mg/mL humanserum albumin, 5.84 mg/mL sodium chloride, 1.164 mg/mL sodium phosphatemonobasic dihydrate, and 2.225 mg/mL sodium phosphate dibasic dihydrate(Formulation No. FD-22512-000-J-45; Product Lot 5C903J; date ofmanufacture, March 1995). The placebo formulation used in these twostudies was a preservative-free phosphate buffered solution containing2.5 mg/mL human serum albumin, 5.84 mg/mL sodium chloride, 1.164 mg/mLsodium phosphate monobasic dihydrate, and 2.225 mg/mL sodium phosphatedibasic dihydrate (Formulation No. FD-22512-000-ABX-45; Product Lot5C902J; date of manufacture, March 1995).

[0226] Both studies were open-label, randomized, placebo controlled,parallel-group, single-center studies. Thirty-two healthy subjects wereenrolled in Clinical Study EPO-PHI-358 and 30 subjects completed thestudy and were included in the PK/PD analyses. Thirty subjects wereenrolled and completed the Clinical Study EPO-PHI-359. Demographic dataof the subjects in these two studies are listed in FIG. 21.

[0227] Frequent serial blood samples were collected for serum EPOdetermination during Weeks 1 and 4 for the 600-IU/kg q.w. dosing regimenand during Week 4 for the 150-IU/kg t.i.w. dosing regimen. Weeklypredose blood samples for serum EPO determination were also collected.For the single dose groups, serial blood samples for serum EPOdetermination were taken over the 4-week study period. Blood sampleswere also collected for determination of percent reticulocytes (% RETI),total red blood cells (RBC), and hemoglobin (HEMO) in blood during the4-week study period.

[0228] Sample analyses for serum EPO were performed at RWJPRI, Raritan,N.J. A RIA kit procedure manufactured by DSL and modified at RWJPRI wasused for the determination of EPO concentrations in serum. This methodand its validation are described in Section 4. Pharmacokinetic andpharmacodynamic parameters were summarized by descriptive statistics.Mean serum EPO concentration-time profiles for subjects in ClinicalStudies EPO-PHI-358 and EPO-PHI-359 are shown in FIGS. 22 and 23,respectively.

[0229] While serum EPO concentrations after single dose administrationsdeclined to the endogenous EPO level by Day 15, the 150-IU/kg t.i.w.dosing regimen was able to maintain serum EPO concentrations above thepre-dose endogenous EPO level throughout the treatment period (FIG. 22).The mean predose endogenous EPO concentration for this group of subjectswas 14±4 mIU/mL, and the mean trough concentrations (corrected forbaseline EPO) before the first, second, and third doses during thefourth dosing week were 19±9, 48±18, and 52±25 mIU/mL, respectively.There was an accumulation of serum EPO during dosing as the C_(max)after the first, second, and the third doses in the last dosing weekranged from 128 to 163, 141 to 214, and 152 to 334 mIU/mL, respectively.The 600-IU/kg once per week dosing regimen maintained serum EPO levelsabove the predose endogenous EPO level up to 5 to 6 days in a dosingweek (FIG. 23). The mean predose endogenous EPO level in these subjectswas 13±3 mIU/mL, and the mean steady-state trough EPO concentration(corrected for baseline EPO) was 11±5 mIU/mL. This dose regimen attaineda higher C_(max) than the 150-IU/kg t.i.w. dosing regimen, although thepredose trough concentrations were near the endogenous EPO level.C_(max) values for the 600-IU/kg q.w. regimen during Weeks 1 and 4ranged from 1203 to 2148 and 920 to 1489 mIU/mL, respectively. Themean±SD pharmacokinetic and pharmacodynamic parameter values are listedin FIG. 24.

[0230] There was a linear relationship between mean C_(max) and dosewith correlation coefficient=0.982 (FIG. 25), suggesting that theabsorption rate of EPO from the injection site was independent of dose.On the other hand, the relationship between AUC and dose was acurvilinear one such that clearance (CL/F) decreased with increasingdoses (FIG. 26).

[0231] Mean percent reticulocyte-time profiles during the 4-week studyperiod are shown in FIG. 27. Percent reticulocytes in blood reachedtheir maximum values on approximately Day 10 after drug administrationfor both single and multiple doses. While the percent reticulocytes inblood after single dose administrations declined to the predose baselinevalues by Day 15, the percent reticulocytes in blood after the twomultiple dosing regimens (150 IU/kg t.i.w. and 600 IU/kg q.w.) weremaintained well above the predose baseline values for up to Day 30. Thisobservation is not unexpected as the normal lifespan of cells in thereticulocyte stage is around 3.5 days in the marrow and 1 to 2 days inthe blood circulation (Hillman, supra). EPO exerts its biologicaleffects by binding to a specific cell-surface receptor on its targeterythroid progenitor cells in bone marrow, the colony-formingunit-erythroid (CFU-E) and the burst-forming unit-erythroid (BFU-E)(Dessypris et al., 1984, Br. J. Haematol., 56:295-306 and Wu et al.,1995, Cell, 83:59-67). These erythroid progenitor cells eventuallymature into reticulocytes which are then released into bloodcirculation. Data from these two studies indicate that reticulocytesproduced from stimulation by a single dose of EPO appeared in the bloodcirculation in about 7 to 10 days. With a lifespan of 1 to 2 days inblood circulation, one would expect reticulocytes produced fromstimulation by a single dose to disappear from blood circulation by Day15. Therefore, a continuous production of reticulocytes requires EPO inserum to be maintained continuously (such as after 150-IU/kg t.i.w.dosing regimen) or intermittently (such as after the 600-IU/kg q.w.dosing regimen) above the endogenous EPO concentration.

[0232] For the single dose data, there was a trend for increase in meanAUC of percent reticulocytes (AUC[UNCORR RETI]) as the mean AUC of EPO[AUC(Day 0-29)] increases (serum EPO concentration corrected for predoseEPO level and AUC value was calculated over a 4-week period). Comparedto single dose data of similar AUC(Day 0-29) values, multiple dose datahave higher mean AUC(UNCORR RETI) values. The data, therefore, suggestthat multiple dosing of EPO is more efficient in stimulating theproduction of reticulocytes than a single dose.

[0233] Despite an EPO AUC-related increase in the production ofreticulocytes, there were no apparent increases in hemoglobin levelsafter single dose administration. The reason for the lack of increase inhemoglobin level after single dosing is not known at this time. On theother hand, both multiple dose regimens were able to deliver a steadyrise in hemoglobin levels, and the patterns of the change in hemoglobinfrom baseline were similar for the two multiple dose regimens (FIG. 28).

[0234] In conclusion, the results from this study show thatpharmacological response to EPO is a function of dose and dosingregimen. The absorption rate of EPO after SC administration wasindependent of dose. Clearance of EPO was dose-dependent, decreasingwith increasing dose. There was an increasing trend of response(AUC[UNCORR RETI]) with AUC(Day 0-29) for single doses. A continuouspharmacological response (a continuous production of reticulocytes andsustained elevation of hemoglobin) requires EPO serum concentration tobe maintained continuously (such as after 150-IU/kg t.i.w. dosingregimen) or intermittently (such as after the 600-IU/kg q.w. dosingregiment) above endogenous EPO levels.

[0235] Clinical Study Epo-PHI-370

[0236] The primary objective of this study was to evaluate thepharmacokinetic profile of EPO after administration of 150 IU/kg t.i.w.or 40,000 IU q.w. and to demonstrate that the two dosing regimensdeliver similar clinical outcomes using hemoglobin as a measure ofclinical effectiveness. Secondary objectives were to assess thepharmacodynamic profiles of EPO after administration of 150 IU/kg t.i.w.or 40,000 IU q.w., and to compare tolerance and safety parametersbetween the two EPO dosing regimens.

[0237] EPO used in this study was formulated as a sterile, colorless,preservative-free, citrated-buffered solution, in single-use vials. TheEPO 10,000 IU/mL (Formula No. FD-22512-000 C-45, Lot D000123) was usedin the 150-IU/kg t.i.w. arm, and the EPO 40,000 IU/mL (Formula No.FD-22512-000 AC-45, Lot D000175) was used in the 40,000-IU q.w. arm.

[0238] This was a single-center, open-label, parallel-design, randomizedstudy conducted in 49 healthy subjects (49 enrolled and analyzed forsafety; 46 completed the study and were analyzed for PK/PD). Subjectswere randomized to two treatment groups and received EPO as either thestandard cancer regimen (150 IU/kg s.c. t.i.w.) or a weekly fixed doseregimen (40,000 IU s.c. q.w.) for four weeks. Blood samples were drawnat predose on Days 1, 8, 15, and during Week 4 for determination ofserum EPO concentrations. Blood samples were also drawn at baseline(Day 1) and at specific time points over the 4-week study period fordetermination of percent reticulocytes, hemoglobin, and total red bloodcell counts.

[0239] Of the 46 subjects who were analyzed for PKJPD, 24 subjects (9males and 15 females) were enrolled in the 150-IU/kg t.i.w. arm and 22subjects (14 males and 8 females) were enrolled in the 40,000-IU q.w.arm. Demographic data and baseline hemoglobin of these subjects arelisted in FIG. 29.

[0240] Sample analyses for serum EPO were performed at PPD Development,Richmond, Va. A RIA kit procedure manufactured by DSL and modified atRWJPRI, was used for the determination of EPO concentrations in serum.

[0241] The sample size of this study was not based on statisticalconsiderations. Summary statistics were provided by treatment group andday for pharmacodynamic parameters; mean, standard deviation, median,range, and standard error were calculated. To compare the PD profiles ofEPO after 150 IU/kg t. i.w. and 40000 IU q.w., analysis of variancemodels appropriate for two way layout were fit to the data, with one ofthe PD parameters of interest (log-transformed AUC of percentreticulocytes, hemoglobin, and total red blood cell counts) as thedependent variable and treatment, gender, and gender by treatment asfixed effects. Since the gender by treatment interaction effect wasfound to be not significant for all three parameters, reduced modelswithout the interaction term were fitted to the data and the treatmentand gender effects were tested at the 5% level using the residual errorterm. The ratio of the means from 40000 IU/week to 150 IU/kg t.i.w. andthe ratio of the means from females to males were estimated using thegeometric least square means obtained from the ANOVA models.

[0242] Mean serum EPO concentration-time profiles (uncorrected forpredose endogenous EPO level) for the 150-IU/kg t.i.w. and the 40,000-IUq.w. groups during Week 4 of the study period are shown in FIG. 30.

[0243] During Week 4 of the 150-IU/kg t.i.w. dosing regimen, EPO peakserum concentrations (corrected for baseline EPO) ranged from 78 to 447mIU/mL (mean C_(max)=191±100 mIU/mL) to trough concentrations of 7.3 to88 mIU/mL (mean trough concentration (C_(min))=39+18 mIU/mL). DuringWeek 4 of the 40,000-IU q.w. dosing regimen, serum EPO peakconcentrations (corrected for baseline EPO) ranged from 197 to 1992mIU/mL (mean C_(max)=785±427 mIU/mL) and were achieved at times rangingfrom 9 to 24 hours (mean t_(max)=18±5 hours), then declinedmulti-exponentially to trough levels ranging from values below thequantification limit of the analytical method (7.8 mIU/mL) to 44 IU/mL(mean trough concentration on Day 29=19±10 mIU/mL) at the end of thedosing week on Day 29. The mean C_(min) over the four week study periodwas 13±9 mIU/mL. The terminal phase of the two dosing regimens seemed tobe in parallel with mean half-life values of 31.8+13.4 (N=13) and39.3±7.1 (N=3) hours for the 150-IU/kg t.i.w. and the 40,000-IU q.w.dosing regimens, respectively.

[0244] Mean (SD) (% CV) pharmacokinetic parameter values are listed inFIG. 31.

[0245] Bioavailability of the 40,000-IU q.w. dosing regimen relative tothat obtained after the 150-IU/kg t.i.w. dose regimen was calculatedusing the following formula:$\frac{{{{AUC}\left( {0\text{-}168} \right)}\quad {of}\quad 40},{000\quad {IU}\quad {q.w.}}}{{{AUC}\left( {0\text{-}168} \right)}\quad {of}\quad 150\quad {IU}\quad \text{/}{kg}\quad {t.i.w.}} \times \frac{450}{\begin{matrix}{40,{000/}} \\{{mean}\quad {body}\quad {weight}}\end{matrix}} \times 100\%$

[0246] Mean body weight was calculated using data from subjects whocompleted the study.

[0247] Bioavailability of EPO after the 40,000-IU q.w. dosing regimenrelative to that after the 150-IU/kg t.i.w. dosing regimen was 176%.

[0248] Linear plots of mean change from baseline versus study day forpercent reticulocytes and hemoglobin concentrations are presented inFIGS. 32 and 33, respectively. Mean pharmacodynamic parameter values(corrected for baseline value) are presented in FIG. 34. The dynamicresponses of the two dosing regimens were similar despite the fact thatserum EPO AUC for the 40,000-IU q.w. dosing regimen was larger than thatfor the 150-IU/kg t.i.w. dosing regimen. There were no statisticallysignificant differences in the AUC of hemoglobin and the AUC of redblood cells between the two dosing regimens, although the AUC of percentreticulocytes after the 40,000-IU q.w. dosing regimen was statisticallylarger (p<0.05) than that after the 150-IU/kg t.i.w. dosing regimen.There were no statistically significant differences in the AUC ofpercent reticulocytes, the AUC of hemoglobin, and the AUC of red bloodcells between male and female subjects.

[0249] The time profiles of changes in hemoglobin and total red bloodcells over the one month study period were similar between the twodosing regimens despite the differences in exposure (i.e. AUC[0-168]) ofEPO in serum and despite a higher production of reticulocytes (asmeasured by area under the curve) for the 40,000-IU q.w. regimen. TheAUCs of hemoglobin and total red blood cell over the one month studyperiod were similar for two dosing regimens. There were no differencesin pharmacodynamic responses for male and female subjects in this study.

[0250] Although the data of this study suggest that the hemoglobinresponses after the 150-IU/kg t.i.w. and the 40,000-IU q.w. dosingregimens were similar and that the two dosing regimens can be usedinterchangeably, there were randomization issues, fluctuating hemoglobinlevels at the study entry, and many subjects were iron-deficient atinitiation (as indicated by transferrin saturation values). Error in therandomization at the study site resulted in an unequal distribution ofmales and females in each treatment group, and contributed to animbalance in mean baseline hemoglobin values. To appropriately confirmthe positive findings of this study, it was decided that a repeat studywould be conducted (Clinical Study EPO-373).

[0251] Clinical Study EPO-PHI-373

[0252] The primary objective of this study was to evaluate the PKprofile of EPO after administration of 150 IU/kg t.i.w. or 40,000 IUq.w. and to demonstrate that the two dosing regimens deliver similarclinical outcomes. Secondary objectives were to assess the PD profilesof EPO after administration of 150 IU/kg t.i.w. or 40,000 IU q.w., andto compare tolerance and safety parameters between the two EPO dosingregimens.

[0253] EPO used in this study was formulated as a sterile, colorless,preservative-free, phosphate-buffered solution, in single-use vials. TheEPO 10,000 IU/mL (Formula No. FD-22512-000-T-45, Lot 99KS077) was usedin the 150-IU/kg t.i.w. arm, and the EPO 40,000 IU/mL (Formula No.FD-22512-000-AA-45, Lot 99KS091) was used in the 40,000-IU q.w. arm.

[0254] This was a single-center, open-label, parallel-design, randomizedstudy conducted in 36 healthy subjects (36 enrolled and analyzed forsafety; 34 completed the study and were analyzed for PK/PD). Subjectswere randomized to two treatment groups and received EPO as either thestandard cancer regimen (150 IU/kg s.c. t.i.w.) or a weekly fixed doseregimen (40,000 IU q.w.) for four weeks. Blood samples were drawn atpredose on Days 1, 8, 15, and during Week 4 for determination of serumEPO concentrations. Blood samples were also drawn at baseline (Day 1)and at specific time points over the 4-week study period fordetermination of percent reticulocytes, hemoglobin, and total red bloodcell counts.

[0255] Of the 34 subjects who were analyzed for PK/PD, 17 subjects (ninemales and eight females) were enrolled in the 150-IU/kg t.i.w. arm and17 subjects (nine males and nine females) were enrolled in the 40,000-IUq.w. arm. Demographic data and baseline hemoglobin of these subjects arelisted in FIG. 35.

[0256] An ELISA kit, manufactured by R&D Systems, Inc. (R&D),Minneapolis, Minn., modified at RWJPRI and cross validated with theoriginal RIA at PPD Development, Richmond, Va., was used for thedetermination of EPO concentrations in serum.

[0257] The sample size of this study was not based on statisticalconsiderations. Summary statistics were provided by treatment group andday for pharmacodynamic parameters; mean, standard deviation, median,range, and standard error were calculated. To compare thepharmacodynamic profiles of EPO after 150 IU/kg t. i. w. and 40000 IUq.w., analysis of variance models appropriate for two way layout werefit to the data, with one of the pharmacodynamic parameters of interest(log-transformed AUC of percent reticulocytes, hemoglobin, and total redblood cell counts) as the dependent variable, and treatment, gender andgender by treatment as fixed effects. Since the gender by treatmentinteraction effect was found to be not significant for all threeparameters, reduced models without the interaction term were fitted tothe data and the treatment and gender effects were tested at the 5%level using the residual error term. The ratio of the means from 40000IU/week to 150 IU/kg t.i.w. and the ratio of the means from females tomales were estimated using the geometric least square means obtainedfrom the ANOVA models.

[0258] Mean serum EPO concentration-time profiles (uncorrected forpredose endogenous EPO level) for the 150-IU/kg t.i.w. and the 40,000-IUq.w. groups during Week 4 of the study period are shown in FIG. 36.

[0259] During Week 4 of the 150-IU/kg t.i.w. dosing regimen, EPOconcentrations in serum (corrected for baseline EPO) ranged from peakconcentrations of 75 to 284 mIU/mL (mean C_(max)=143±54 mIU/mL) totrough level values ranging from values below the quantification limitof the analytical method (7.8 mIU/mL) to 40 IU/mL (mean troughconcentration (C_(min))=18±9 mIU/mL). During Week 4 of the 40,000-IUq.w. dosing regimen, serum EPO concentrations (corrected for baselineEPO) reached peak concentrations (mean C_(max)=861+445 mIU/mL) at timesranging from 1 to 24 hours (mean t_(max)=16±8 hours), then declinedmulti-exponentially to trough level values ranging from values below thequantification limit of the analytical method (7.8 mIU/mL) to 5.9 mIU/mL(mean trough concentration on Day 29=2.0±1.5 mIU/mL) at the end of thedosing week on Day 29. Mean C_(min) of the 40,000 IU q.w. during the4-week study period was 3.8±4.3 mIU/mL. The terminal phase of the twodosing regimens seemed to be in parallel with mean half-life values of19.4±8.1 hours (n=9) and 15.0±6.1 hours (n=9) for the 150-IU/kg t.i.w.and the 40,000-IU q.w. dosing regimens, respectively.

[0260] Mean (SD) (% CV) pharmacokinetic parameter values are listed inFIG. 37.

[0261] Bioavailability of the 40,000-IU q.w. dosing regimen relative tothat obtained after the 150-IU/kg t.i.w. dose regimen was calculatedusing the following formula:$\frac{{{{AUC}\left( {0\text{-}168} \right)}\quad {of}\quad 40},{000\quad {IU}\quad {q.w.}}}{{{AUC}\left( {0\text{-}168} \right)}\quad {of}\quad 150\quad {IU}\quad \text{/}{kg}\quad {t.i.w.}} \times \frac{450}{\begin{matrix}{40,{000/}} \\{{mean}\quad {body}\quad {weight}}\end{matrix}} \times 100\%$

[0262] Mean body weight was calculated using data from subjects whocompleted the study.

[0263] Bioavailability of EPO after the 40,000-IU q.w. dosing regimenrelative to that after the 150-IU/kg t.i.w. dosing regimen was 239%.

[0264] Linear plots of mean change from baseline versus study day forpercent reticulocytes, hemoglobin concentrations, and total red bloodcell counts are presented in FIGS. 38, 39, and 40, respectively.

[0265] Mean pharmacodynamic parameter values (corrected for baselinevalue) are presented in FIG. 41. The dynamic responses of the two dosingregimens were similar despite the fact that serum EPO AUC for the40,000-IU q.w. dosing regimen was larger than that for the 150-IU/kgt.i.w. dosing regimen. There were no statistically significantdifferences in the AUC of percent reticulocytes, AUC of hemoglobin, andAUC of red blood cells between the two dosing regimens. There were nostatistically significant differences (p>0.05) in the AUC of percentreticulocytes between male and female subjects. However, the AUC ofhemoglobin and the AUC of red blood cells were statistically (p=0.038and 0.042, respectively) larger in females than in males. Thesedifferences were not clinically significant.

[0266] The time profiles of changes in percent reticulocytes,hemoglobin, and total red blood cells over the one month study periodwere similar between the two dosing regimens despite the differences inexposure of EPO in serum (in terms of AUC[0-168]). In addition, therewere no statistically significant differences (p>0.05) in AUC of percentreticulocytes, AUC of hemoglobin, and AUC of total red blood cells overthe one month study period between the two dosing regimens. There wereno differences in pharmacodynamic responses between male and femalesubjects in this study. The data of this study indicate that thehemoglobin responses after the 150-IU/kg t.i.w. and the 40,000-IU q.w.dosing regimens were similar, thereby justifying that the two dosingregimens can be used interchangeably.

[0267] In conclusion, there was an expected difference in total exposureof EPO in serum after the 150-IU/kg t.i.w. and the 40,000-IU q.w. dosingregimens. The hemoglobin responses were similar, thereby justifying thatthe two dosing regimens can be used interchangeably. Thus, the presentstudies show that a once-weekly EPO dosing regimen can be used. Thisregimen overcomes the disadvantages associated with the commonly useddosing regimens. In a preferred embodment, a 40,000 IU dose iscontemplated based upon these clinical studies. The present inventionalso contemplates a once every two weeks dosing regimen, e.g., at a EPOdose of 80,000 to 120,000 IU. In a specific embodiment, a once every twoweeks dose of EPO is used.

[0268] Results

[0269] Pharmacokinetics After IV Administration

[0270] After IV administration in healthy volunteers or patients withimpaired renal function, r-HuEPO is distributed in a volume comparableto the plasma volume, and plasma concentrations decay with meanhalf-life values ranging from 4 to 11.2 hours which have been reportedto be shortened after repeated administrations (Macdougall et al., 1991,Clin. Pharmacokinet., 20:99-113). There were dose-proportional increasesin C_(max) and AUC values following single intravenous doses of 50 to1000 IU/kg in healthy subjects (Flaharty et al., 1990, Clin. Pharmacol.Ther., 47:557-64), and clearance (CL) was reported to be independent ofdose after single bolus intravenous administration of 10-, 100-, and500-IU/kg doses in healthy subjects, with respective mean values of5.89±1.53, 7.02±1.14, and 6.88±1.19 mL/h/kg (Veng-Pendersen et al.,1995, J. Pharma. Sci., 84(6):760-767). However, clearance was alsoreported to be higher after a single intravenous 10-IU/kg dose (13.1mL/h/kg) than after that after single intravenous doses of 100 and 500IU/kg in healthy subjects (respectively CL=7.9 and 6.2 mL/h/kg) (Widnesset al., 1996, J. Appl. Physiol., 80(1):140-148).

[0271] Pharmacokinetics after SC Administration

[0272] A summary of pharmacokinetic parameters after SC administrationfrom Clinical Studies EPO-PHI-358, EPO-PHI-359, EPO-PHI-370, andEPO-PHI-373 is given in FIG. 42.

[0273] After single or weekly SC administration, serum EPOconcentrations reached maximum value in times (t_(max)) ranging from 9to 36 hours. The mean t_(max) was similar for different single doses andranged from 15.6+5.8 to 28.8±7.8 hours. There was a linear relationshipbetween mean C_(max) and dose (correlation=0.982), suggesting that theabsorption rate of EPO from the injection site was independent of dose.On the other hand, the relationship between AUC and dose was acurvilinear one such that clearance (CL/F) decreased with increasingdoses (Cheung et al., 1998, Clin. Pharmacol. Ther., 64:412-423). Datafrom Clinical Studies EPO-PHI-373 and EPO-PHI-370 indicate that the150-IU/kg t.i.w. dosing regimen had a higher CL/F value than the40,000-IU q.w. weekly dosing regimen and data in Clinical StudiesEPO-PHI-358 and EPO-PHI-359 indicate that the 150-IU/kg t.i.w. dosingregimen had a higher CL/F value than the 600-IU/kg q.w. dosing regimen.

[0274] As indicated in FIG. 42, EPO decays at a much slower rate afterSC administration than after IV administration. Half-life values rangedfrom 15.9 to 221 hours after single SC doses of 300 to 2400 OU/kg, andmean half-life values after the 150-IU/kg t.i.w. and 40,000-IU q.w.dosing regimens were 19.4±8.1 and 15.0±6.1 hours, respectively. Thehalf-life values were independent of dose in the dose range studied. Thelonger half-life value observed after SC administration compared to IVadministration is probably a reflection of the absorption half-life fromsubcutaneous tissues.

[0275] While serum EPO concentrations after single dose administrationsdeclined to the endogenous EPO level by Day 15 (earlier for the lowerdoses), the 150-IU/kg t.i.w. dosing regimen was able to maintain serumEPO concentrations above the predose endogenous EPO level throughout thetreatment period (Cheung, supra). The weekly dosing regimens of 600IU/kg/mL and 40,000 IU q.w. maintained serum EPO levels above thepredose endogenous EPO level up to 5 to 6 days in a dosing week (Cheung,supra). These weekly dosing regimens attained a higher C_(max) than the150-IU/kg t.i.w. dosing regimen, although the predose troughconcentrations were near the endogenous EPO level.

[0276] Pharmacodynamics of EPO after SC Administration

[0277] After single (300 to 2400 IU/kg) or multiple (150 IU/kg t.i.w.,600 IU/kg q.w., or 40,000 IU q.w.) SC administrations of EPO, percentreticulocytes began to increase by Days 3 to 4. Percent reticulocytesafter single dose administrations reached their maximum values at Days 6to 12 (Cheung, supra), whereas percent reticulocytes after multiple doseregimens reached peak values at times ranged from Day 8 to the lastblood sample point on Day 29 (Cheung, supra). All multiple dose regimensstimulated modest, but sustained increases in percent reticulocytes(approximately 2 to 7%) which were maintained above the predose baselinevalues through Days 22 to 29, while the percent reticulocytes aftersingle dose administrations declined to baseline values by Days 15 to 22(Cheung, supra).

[0278] The relationship between mean AUC of the change (from baseline)in percent reticulocyte [AUC(RETI)] and mean AUC of EPO (corrected forpredose endogenous EPO level) [AUC(Day 0-29)] is shown in FIG. 43 fordata in Clinical Studies EPO-PHI-358, EPO-PHI-359, and in FIG. 44 fordata in Clinical Studies EPO-PHI-370 and EPO-PHI-373. The AUC values forboth change in percent reticulocyte from baseline and EPO werecalculated over a four-week period. The AUC(RETI) values in ClinicalStudies EPO-PHI-358 and EPO-PHI-359 can not be compared to those inClinical Studies EPO-PHI-370 and EPO-PHI-373 as the times of samplecollection for reticulocytes were different. The sampling schedule wasless frequent in Clinical Studies EPO-PHI-358 and EPO-PHI-359, and theAUC(RETI) values could have been underestimated. Data from ClinicalStudies EPO-PHI-358 and EPO-PHI-359 indicated that, for the single dosedata, there was a trend of increase in mean AUC(RETI) as the meanAUC(Day 0-29) of EPO increases. For the multiple dose regimens based ondata from Clinical Studies EPO-PHI-370 and EPO-PHI-373, there is also atrend of increase in mean AUC(RETI) as the mean AUC(Day 0-29) of EPOincreases.

[0279] The total EPO administered in one month for the 150-IU/kg t.i.w.regimen was 1800 IU/kg, and for the 600-IU/kg q.w. regimen wasapproximately 2400 IU/kg. Although the 150-IU/kg t.i.w. regimen had amuch smaller EPO AUC(Day 0-29) value than the 1800-IU/kg single doseregimen, they had similar mean AUC(RETI) values. Similarly, the600-IU/kg q.w. regimen had a much smaller EPO AUC(Day 0-29) value thanthe 2400-IU/kg single dose regimen, but they also had similar meanAUC(RETI) values. Thus, EPO (per unit AUC exposure) after multipledosing is more efficient in producing reticulocytes than after a singledose.

[0280] Despite an EPO AUC-related increase in the production ofreticulocytes, there were no apparent increases in hemoglobin aftersingle dose administration. On the other hand, multiple dose regimenswere able to deliver a steady rise in hemoglobin, and the patterns ofthe rise in hemoglobin were similar between the 150-IU/kg t.i.w. and40,000-IU q.w. dosing regimens as demonstrated in the pivotal ClinicalStudy EPO-PHI-373 (FIG. 45) and the supportive Clinical StudyEPO-PHI-370 (FIG. 46), and were similar between the 150-IU/kg t.i.w. and600-IU/kg q.w. regimens as demonstrated in the pilot Clinical StudiesEPO-PHI-358 and EPO-PHI-359. The lack of hemoglobin response aftersingle dosing is not known at this time. There are two possibleexplanations for this: the increase in reticulocytes after single doses(maximum increase of percent reticulocytes after 2400-IU/kg singledose=6.6%) might not have been sustained for long enough to lead to anysubstantial increase in hemoglobin; and it has been hypothesized thatthe survival of young red blood cells depends on the continuous presenceof EPO in the blood circulation (Alfrey et al., 1997, The Lancet,349:1389-1390). After single dose administration, reticulocytes peakedat times ranging from 6 to 12 days and returned to predose levels attimes up to 15 days. The lifespan of cells in reticulocyte stage isabout 1 to 2 days in the blood circulation (Hillman et al., 1967, SemHaematol., 4(4):327-336).

[0281] Therefore, one would expect young red cells to appear in theblood circulation about 7 to 14 days after dosing. By then, EPOconcentrations after single dose administration were at the endogenouslevel, which may have resulted in death of the young red cells due tothe absence of sufficient EPO in the blood circulation to sustain theirsurvival. On the other hand, EPO concentrations were maintained abovethe endogenous level continuously after the t.i.w. dosing regimen orintermittently after the weekly dosing regimen, and therefore thesurvival of young red cells was sustained, leading to a continuous risein hemoglobin during the study period.

[0282] Consequently, to sustain the survival of a large number ofpharmacologically produced young red cells, serum EPO concentrationshave to be above the endogenous level.

[0283] Conclusion

[0284] In conclusion, data from these four Phase I studies in healthysubjects indicate that the pharmacokinetics and pharmacodynamics of EPOin humans are nonlinear after SC administration. The data from thesestudies also clearly demonstrate the 150-IU/kg t.i.w. dosing regimendelivered similar hemoglobin response as the 40,000-IU per week dosingregimen, thereby justifying that the two dosing regimens can be usedinterchangeably. Accordingly, EPO is administered about 40,000 IU/kgonce a week for two consecutive weeks. The first dose of EPO facilitatesthe production of reticulocytes from RBC progenitor cells. The seconddose of EPO is administered to coincide with the reticulocytepharmacodynamic profile of the patient. The second dose of EPO will beadministered 6-10 days following the initial dose, and preferably at thetime when the reticulocyte concentration peaks following the first EPOdose.

Example 2 Evaluation of EPO PK/PD Profile After Administration of 150IU/kg t.i.w. and 40,000 IU q.w.

[0285] In specific indications, such as cancer, subjects are treatedwith 150 IU/kg epoetin alfa t.i.w. Thus, it remains an important goal tochange the currently approved dosing schedule to a more convenient(i.e., once per week or once every two weeks) dosing schedule andregimen. A less frequent administration will improve user acceptance andconvenience.

[0286] The pharmacokinetic and pharmacodynamic properties of themultiple dosing regimen of epoetin alfa have been defined in theprevious example (EPO-PHI-358 and EPO-PHI-359). The data suggest that150 IU/kg t.i.w. and 600 IU/kg/week dosing regimens have similarpharmacodynamic responses (e.g., a rise in hemoglobin). Therefore,epoetin alfa can potentially be administered as a weekly per kilogramdose. Since 600 IU/kg is equivalent to 42,000 IU for a 70-kg person, thepresent study was conducted to demonstrate that a fixed dosing regimenof 40,000 IU per week delivered a comparable pharmacodynamic response asthe approved dosing regimen of 150 IU/kg t.i.w.

[0287] The primary objective of this study was to evaluate thepharmacokinetic profile of epoetin alfa after administration of 150IU/kg t.i.w. or 40,000 IU q.w., and to demonstrate that the two dosingregimens deliver similar clinical outcomes.

[0288] The secondary objectives were to assess the pharmacodynamicprofiles of epoetin alfa after administration of 150 IU/kg t.i.w. or40,000 IU q.w., and to compare tolerance and safety parameters betweenthe two epoetin alfa dosing regimens.

[0289] Thirty-six healthy adult volunteers (18 males and 18 females)were enrolled into this open-label, parallel-design, randomized,single-center study. Subjects were to be between the ages of 18 and 45years old with hemoglobin levels between 12.0 and 14.0 g/dL, inclusive,for females and between 13.0 and 14.0 g/dL, inclusive, for males.Subjects were screened for study eligibility based on inclusion andexclusion criteria and randomized to one of two treatment groups. Group1 received the standard cancer regimen of 150 IU/kg of SC EPO, t.i.w.for four weeks. Group 2 received a weekly fixed dose regimen of 40,000IU epoetin alfa s.c. q.w. for four weeks.

[0290] Subjects were to receive daily oral iron supplementation duringthe study (two capsules of Ferro-Grad® each containing 105 mg ofelemental iron).

[0291] Blood samples were drawn at baseline and at specific time pointsduring the study for the determination of serum erythropoietinconcentrations, complete blood count (CBC), including percentreticulocytes, hemoglobin concentrations, and hematocrit values. Safetyevaluations were based on the incidence and type of treatment-emergentadverse events, changes in clinical laboratory tests (hematology andchemistry), vital sign measurements, and physical examination results.In addition, serum iron, calculated transferrin saturation, and ferritinconcentrations were monitored during the study.

[0292] Safety was based on the incidence and severity oftreatment-emergent adverse events, and on changes from prestudy inphysical examination findings, vital sign measurements, and clinicallaboratory parameters.

[0293] The protocol was amended after all subjects were initiated. Theamendment clarified inclusion and exclusion criteria, concomitanttherapy, laboratory parameters, urinalysis, vital sign measurements, andthe dosing regimen for the 40,000 IU q.w. group.

[0294] Three subjects randomized to the 150 IU/kg t.i.w. group and onesubject in the 40,000 IU q.w. group had hemoglobin entry criteriamarginally above the limit specified in the inclusion/exclusion criteria(14.0 g/dL), but were included in the study. Subjects 2012, 2015, and2016 in the 150 IU/kg t.i.w. group had screening hemoglobins of 14.1g/dL; subject 2018 in the 40,000 IU q.w. group had a screeninghemoglobin of 14.2 g/dL. Four subjects in the 40,000 IU q.w. group hadscreening ferritin values below the limit of 45 ng/mL specified in theexclusion criteria: subject 1006 had a screening value of 40 ng/mL,subject 1010 had a screening value of 43 ng/mL, and subjects 1015 and2011 had screening values of 44 ng/mL. One subject in each treatmentgroup (subject 2015 in the 150 IU/kg group and subject 2014 in the40,000 IU group) had screening transferrin saturation values of 19%,slightly below the inclusion criteria of >20%. Subject 2002 (40,000 IU)weighed 0.1 kg above the maximum value allowed by protocol inclusioncriteria for someone his height. Additionally, subject 2016 (150 IU/kgt.i.w.) took herbal sleeping tablets four days prior to initiation ofepoetin alfa therapy.

[0295] Study Drug Information

[0296] Epoetin alfa was formulated as a sterile, colorless,preservative-free, phosphate-buffered solution, supplied in single-usevials. Commercial product was used in this study, and it wascommercially labeled. The epoetin alfa 10,000 IU/mL solution was FormulaNo. FD 22512-000-T-45, Lot No. 99KS077, and the epoetin alfa 40,000IU/mL solution was Formula No. FD 22512-000-AA-45, Lot No. 99KS091.

[0297] Dosage and Administration

[0298] Subjects were admitted to the investigator's facility at least 12hours prior to the administration of study medication on Day 1. Subjectswere fasted for at least ten hours prior to dosing on Day 1; water wasavailable ad libitum. Subjects were randomly assigned and received oneof two study medications as follows: Group Treatment Group 1 StandardCancer Regimen (N = 18) 150 IU/kg of epoetin alfa s.c. t.i.w. for fourweeks Group 2 Weekly Fixed Dose Regimen (N = 18) 40,000 IU of epoetinalfa s.c. q.w. for four weeks

[0299] If, at any time during the treatment phase, the hemoglobin forany subject equalled or exceeded 18.0 g/dL, a second sample was drawn toconfirm the first finding. If confirmed, phlebotomy was done to reducethe hemoglobin level; initially, one unit of blood was to be removed.The hemoglobin was measured again once the subject was stabilized. Ifthe hemoglobin level was still 18.0 g/dL or greater, another 0.5 to oneunit of blood was to be removed, and the hemoglobin measured. Anyphlebotomized subject was discontinued from epoetin alfa therapy andunderwent the required completion evaluations and procedures.

[0300] The 10,000 IU/mL formulation of epoetin alfa was used for Group 1(Standard Cancer Regimen), and the 40,000 IU/mL formulation of epoetinalfa was used for Group 2 (Weekly Fixed Dose Regimen).

[0301] Concomitant Therapy

[0302] Subjects were instructed to take no medications (prescription,over-the-counter (OTC), herbal, or “natural”) beginning two weeks priorto the first dose of study drug and thereafter for the entire durationof the study. In case of headache or flu-like symptoms, paracetamolcould be administered. If the administration of any medication becamenecessary, it was to be reported on the appropriate case report form(CRF) and source document.

[0303] Subjects received daily oral iron supplementation during thestudy (two capsules of Ferro-Grad® each containing 105 mg of elementaliron.

[0304] Study Evaluations

[0305] Time and Events Schedule

[0306] The study was divided into three phases: screening, treatment,and completion/early withdrawal. Subjects were evaluated for theireligibility during the screening period (procedures performed within twoweeks of study drug administration). Subjects were randomly assigned toone of the two treatment groups and then entered the treatment phase.Subjects were confined within the clinic at least 12 hours prior to theadministration of study drug on Day 1, and remained confined for atleast 24 hours after dosing, until all tests were performed. Subjectswere fasted at least ten hours prior to dosing on Day 1, but receivedwater ad libitum. The treatment phase consisted of study drugadministration (dosing on Days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24,and 26 for the 150 IU/kg t.i.w. group and on Days 1, 8, 15, and 22 forthe 40,000 IU q.w. group). On Day 22, a second confinement period beganwith the administration of epoetin alfa and continued for at least 144hours post-dosing. Evaluations of pharmacokinetic, pharmacodynamic, andsafety parameters were performed on all subjects at periodic intervalsduring the 28-day treatment phase. Study completion evaluations andprocedures were performed on Day 29, or upon early withdrawal from thestudy.

[0307] Pharmacokinetic Evaluations

[0308] Sample Collection and Handling

[0309] Venous blood samples, 2.5 mL each, were collected by directvenipuncture into vacuum tubes for the determination of serumerythropoietin concentration from the 150 IU/kg epoetin alfa t.i.w.group (Group 1) at the following time points:

[0310] Day 1: 30, 20, and 10 minutes prior to the initial dose of studymedication.

[0311] Days 8 and 15: immediately prior to dosing.

[0312] Days 22 and 24: immediately prior to dosing and at 0.5, 3, 6, 9,12, 15, 18, 24, 30, and 36 hours post-dosing.

[0313] Day 26: immediately prior to dosing and at 0.5, 3, 6, 9, 12, 15,18, 24, 36, 48 and 72 hours post-dosing.

[0314] Blood samples for the determination of serum erythropoietinconcentration from the 40,000 IU q.w. group (Group 2) at the followingtime points:

[0315] Day 1: 30, 20, and 10 minutes prior to the initial dose of studymedication.

[0316] Days 8 and 15: immediately prior to dosing.

[0317] Days 22-28: immediately prior to dosing and at 0.5, 3, 6, 9, 12,15, 18, 24, 30, 36, 48, 48.5, 51, 54, 57, 60, 63, 66, 72, 78, 84, 96,96.5, 99, 102, 105, 108, 111, 114, 120, 126, 132, 144, and 168 hourspost Day 22 epoetin alfa administration.

[0318] Samples were allowed to clot at room temperature forapproximately 20 minutes and then centrifuged for ten minutes at 1200rpm in a refrigerated centrifuge. Serum was dispensed into a prelabeledpolypropylene vial. The samples were frozen at −20 C and were storedfrozen at this temperature until analyzed.

[0319] Analytical Procedures

[0320] Sample analyses for serum epoetin alfa were performed at PPDdevelopment, Richmond, Va. An enzyme-linked immunosorbent assay (ELISA)kit procedure manufactured by R&D Systems, Inc., (R&D), Minneapolis,Minn., and modified at RWJPRI, was used for the determination oferythropoietin concentrations in serum. The commercially available ELISAis a direct, double-antibody sandwich assay. Microtiter wells, precoatedwith a mouse monoclonal antibody specific for rHuEPO, are used tocapture EPO. The bound EPO is labeled with anti-EPO polyclonal (rabbit)antibody and horseradish peroxidase. An optical signal is produced withthe addition of substrate. The major in-house modification of the R&Dkit was use of in-house recombinant human erythropoietin in standardsand spiked quality control samples.

[0321] Standard concentrations used in the assay were 7.8, 15.6, 31.3,50, 62.5, 100, 125 and 250 mIU/mL. Sensitivity, defined as the loweststandard giving acceptable precision, was 7.8 mIU/mL and the assay rangewas extended to 5,000 mIU/mL via quality control dilutions.

[0322] Pharmacokinetic Parameters

[0323] Serum concentrations of erythropoietin were measured. Thepharmacokinetic parameters C_(max), t_(max), C_(min), AUC₍₀₋₁₆₈₎, CL/F,and t_(1/2) were measured by model-independent methods.

[0324] Pharmacodynamic Analysis

[0325] Pharmacodynamic parameters included measurement of changes frombaseline in percent reticulocytes, red blood cells, and hemoglobinconcentrations and their relationship to serum erythropoietinconcentrations.

[0326] Safety Evaluations

[0327] Adverse Events

[0328] Treatment-emergent adverse events were defined as any noxious orunintended events observed during clinical investigation that were newin onset or aggravated in severity or frequency, including pathologicfindings that required medical intervention, including additionaldiagnostic procedures or alteration of study therapy.

[0329] Each subject was observed throughout the study beginning with thefirst dose for possible adverse events. Adverse event reports wereidentified by voluntary subject reporting. The investigator recorded onthe subject's CRF any treatment-emergent adverse events regardless oftheir relationship to study drug. Adverse events were characterizedaccording to date of onset, severity (marked, moderate, or mild),relationship to study drug (very likely, probable, possible, doubtful,or not related), action taken regarding study therapy (none, dosereduced, drug stopped temporarily, or drug stopped permanently), andwhether or not the event was serious. Information on concomitant therapyand outcome was also recorded.

[0330] Serious adverse events were defined as those that were fatal orimmediately life-threatening, required or prolonged inpatienthospitalization, caused persistent or significant disability orincapacity, or were congenital anomalies, birth defects, or overdoses.The investigator was instructed to report all serious adverse eventsimmediately to RWJPRI. The investigator was to collect information onserious adverse events for up to 35 days after the last assessment onDay 29. Safety data were also reviewed for potentially serious adverseevents; these were considered to be those that were sufficiently severeor alarming to require medical intervention.

[0331] Clinical Laboratory Tests

[0332] Sample analyses for clinical laboratory tests were performed atHavenfern Laboratories, Berks, UK. Blood samples, including hemoglobin,hematocrit, percent reticulocytes, red blood cells (RBC), meancorpuscular volume (MCV), mean corpuscular hemoglobin (MCH), meancorpuscular hemoglobin concentration (MCHC), and platelets were drawnduring screening and on Days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26,and 29, between 8 and 10 AM, if possible. The screening CBC alsoincluded total erythrocyte count and total leukocyte count withdifferential.

[0333] Serum chemistry samples were drawn at screening and termination(Day 29); parameters included glucose, calcium, sodium, potassium,chloride, phosphorus, blood urea nitrogen (BUN), total bilirubin,creatinine, total protein, cholesterol, albumin, uric acid, alkalinephosphatase, serum glutamic-oxaloacetic transaminase (SGOT; aspartateaminotransferase (AST), serum glutamic-pyruvic transaminase (SGPT;alanine aminotransferase (ALT), and lactic dehydrogenase (LDH).

[0334] Blood samples for iron parameters (serum iron, total iron bindingcapacity (TIBC), and ferritin levels) were drawn during screening and onDays 8, 15, 22, and

[0335] 29. Transferrin saturation was calculated as iron/total ironbinding capacity.

[0336] Urine testing was performed via dipstick. If the blood orleukocyte esterase was positive (1+ or greater) and/or protein ornitrate was trace or greater, a urine specimen was to be sent to thecentral laboratory for microscopic examination.

[0337] Other Safety Observations

[0338] Vital Signs

[0339] Vital sign measurements of sitting blood pressure, pulse rate,and oral temperature were recorded at the screening visit, prior tostudy drug administration on Days 1, 8, 15, and 22, and on Day 29(termination). Respiration rate and body weight measurements were takenat screening and termination; height measurement was obtained only atthe screening visit.

[0340] Physical Examinations

[0341] Physical examinations were performed at the screening visit andon Day 29.

[0342] Data Quality Assurance

[0343] Before the study site was selected, the investigator, study sitepersonnel, and facility were evaluated by clinical RWJPRI personnel. Theprotocol and statement of informed consent were reviewed and approved bythe investigator's Ethics Committee (EC) before initiation of the study.Case report forms were reviewed for accuracy and completeness by RWJPRIpersonnel during the periodic on-site monitoring visits.

[0344] Discrepancies in the data were resolved with the investigator ordesignees. The data were entered into the RWJPRI data base andappropriate computer edit programs were run to verify the accuracy ofthe data base.

[0345] Statistical Methods

[0346] The sample size of this study was not based on statisticalconsiderations. Therefore, the analysis is descriptive; no statisticaltests were performed on pharmacokinetic parameters. Summary statistics,including mean, standard deviation, median, and range were provided bytreatment group for hematology, serum chemistry, and vital signmeasurements.

[0347] The primary objectives (i.e., to evaluate the pharmacokineticprofile of epoetin alfa after administration of 150 IU/kg t.i.w. or40,000 IU q.w., and to demonstrate that the two dosing regimens deliversimilar clinical outcomes, using hemoglobin as a measure of clinicaleffectiveness) was addressed by descriptive comparison ofpharmacokinetic parameters obtained after study drug administration.

[0348] Pharmacokinetics

[0349] The following pharmacokinetic parameters were calculated by modelindependent methods using the WinNonlin software, Version 1.1(Scientific Consulting, Incorporation, Apex, N.C.):

[0350] Peak serum concentration (C_(max)): the observed maximum serumconcentration during the fourth week of the dosing period for the 150IU/kg t.i.w. dosing regimen and the 40,000 IU q.w. dosing regimen.

[0351] Time to C_(max) (t_(max)): the time at which C_(max) occurs.T_(max) was not reported for the epoetin alfa t.i.w. treatment groupbecause C_(max) occurred randomly at any one of the three doses duringthe fourth dosing week.

[0352] Mean predose trough concentration (C_(min)). C_(min) for the 150IU/kg t.i.w. regimen was estimated by averaging the predose troughconcentrations on Days 22, 24, and 26, and the concentration at 72 hoursafter the last dose on Day 26. C_(min) for the 40,000 IU q.w. regimenwas estimated by averaging the predose concentration on Days 8, 15, 22,and the concentration at 168 hours after the last dose on Day 22.

[0353] Area under the serum concentration-time curve from time zero tothe last blood sampling time AUC₍₀₋₁₆₈₎ during the last dosing week forepoetin alfa 40,000 IU q.w. and 150 IU/kg t.i.w.: calculated using thelinear trapezoidal rule.

[0354] Clearance after SC administration (CL/F): calculated by dividingdose (per kg) by AUC_((0-168).)

[0355] Terminal elimination half-life (t_(1/2)): computed from0.693/elimination rate constant. The elimination rate constant wasestimated by linear regression of consecutive data points in theterminal linear region of the log-linear concentration-time plot. Aminimum of three data points were used in the regression. The t_(1/2)values were not reported for those regressions with a correlationcoefficient (r) less than 0.975 (or r²<0.95).

[0356] The mean, standard deviation, and coefficient of variation of thepharmacokinetic parameters were calculated for each treatment.

[0357] The pharmacokinetic parameters were calculated using serumerythropoietin concentrations corrected for predose endogenouserythropoietin levels. Postdose serum concentration values werecorrected for predose baseline erythropoietin concentrations bysubtracting from each of the values the mean baseline erythropoietinconcentration determined from the samples collected at 30, 20, and 10minutes before dosing. Predose serum erythropoietin concentrations werenot included in the calculation of mean value if they were below thequantification limit of the assay. If the concentration values of allthree of a subject's predose samples were below the quantification limitof the assay, then the quantification limit of the assay, 7.8 mIU/mL wasassigned as the mean baseline erythropoietin concentration for thatsubject. Actual blood drawn times (in hour relative to the time of thefirst dose) were used in the calculation of pharmacokinetic parameters.

[0358] Bioavailability of the 40,000 IU q.w. dosing regimen relative tothat obtained after the 150 IU/kg t.i.w. dose regimen was calculatedusing the following formula:$\frac{{{AUC}_{({0\text{-}168})}\quad {of}\quad 40},{000\quad {IU}\quad {q.w.}}}{{AUC}_{({0\text{-}168})}\quad {of}\quad 150\quad {IU}\quad \text{/}{kg}\quad {t.i.w.}} \times \frac{450}{\begin{matrix}{40,{000/}} \\{{mean}\quad {body}\quad {weight}^{*}}\end{matrix}} \times 100\%$ ^(*)  Mean  body  weight  of  subjects  in  the  40, 000  IU  q.w.  group  who  completed  the  study

[0359] Pharmacodynamics

[0360] Summary statistics including mean, standard deviation, median,range, and standard error were provided by treatment group and day forpercent reticulocytes and hemoglobin concentrations. Calculations werebased on subjects who completed the study. Baseline, Days 1, 3, 5, 8,10, 12, 15, 17, 19, 22, 24, 26, and 29 were summarized; “windowing” wasperformed to include laboratory test results in the summaries if asubject did not have data collected at the day(s) specified. Changesfrom baseline were summarized by treatment group and day. Linear plotsof mean change from baseline values versus study day were generated forreticulocytes, hemoglobin, and ferritin.

[0361] Pharmacokinetics/Pharmacodynamics

[0362] Nominal times (in day relative to the first dose on Day 1) asstated in the protocol were used in the calculation of pharmacodynamicparameters. The following pharmacodynamic parameters were calculated bymodel independent methods using the WinNonlin software, Version 1.1(Scientific Consulting, Incorporation, Apex, N.C.):

[0363] Area under the serum concentration-time curve of percentreticulocytes from time zero to 672 hours (Day 29) post-initiation ofdosing [AUC(RETI)] was calculated using the linear trapezoidal rule.AUC(RETI) was estimated using percent reticulocyte values corrected forpredose percent reticulocyte value. The mean of the −10 minute and −30minute predose measurements was used as the predose percent reticulocytevalue. For those corrected percent reticulocyte data with negativevalues, a zero value was used in the estimation of AUC(RETI).

[0364] Area under the serum concentration-time curve of hemoglobin fromtime zero to 672 hours (Day 29) post-initiation of dosing [AUC(HEMO)]was calculated using the linear trapezoidal rule. AUC(HEMO) wasestimated using hemoglobin values corrected for predose hemoglobinvalue. The mean of the −10 minute and −30 minute predose measurementswas used as the predose hemoglobin. For those corrected hemoglobin datawith negative values, a zero value was used in the estimation ofAUC(HEMO).

[0365] Area under the serum concentration-time curve of total red bloodcell count (RBC) from time zero to 672 hours (Day 29) post-initiation ofdosing [AUC(RBC)] was calculated using the linear trapezoidal rule.AUC(RBC) was estimated using RBC values corrected for predose RBC value.The mean of the −10 minute and −30 minute predose measurements was usedas the predose RBC. For those corrected RBC data with negative values, azero value was used in the estimation of AUC(RBC).

[0366] The 40,000 IU q.w. to 150 IU/kg t.i.w. ratios of AUC(RETI),AUC(HEMO), and AUC(RBC) were determined.

[0367] Safety

[0368] The safety evaluations were based upon the type and incidence ofadverse events reported by the subjects and changes in physicalexaminations, clinical laboratory data, and vital signs.Treatment-emergent adverse events were classified by body system,preferred term, and included term. Adverse events were coded inaccordance with the World Health Organization Adverse ReactionTerminology (WHOART) dictionary where the included term is thedescription most closely related to the investigator's terminology, thepreferred term is a group of closely related included terms, and thebody system is a broad category including related preferred terms.Treatment-emergent adverse events were summarized by body system andpreferred term and presented as individual subject data listings.

[0369] Clinical laboratory data and vital signs were summarized andpresented as individual subject data listings.

[0370] Data Storage

[0371] The protocol, report, and raw data from this study are stored inthe Document Management, Information Management department of RWJPRI.The data can be found in the project notebook maintained for DrugMetabolism Study DM00009.

[0372] Study Duration

[0373] Dosing and serum sample collection for both groups were conductedduring the period from 21 Feb. 2000 through 29 Mar. 2000. Validation ofthe erythropoietin serum assay occurred between 21 Jan. 2000 and 3 Feb.2000. Serum samples were analyzed during the period from 24 Mar. 2000through 4 Apr. 2000.

[0374] Results

[0375] Demographic and Baseline Characteristics

[0376] Thirty-six healthy adults (18 subjects per group) were enrolledin this study and were randomly assigned to one of two treatment groups.Overall, the majority of subjects (89%) were white, and the mean age was26.5 years (range 18-41 years). The mean body weight of subjects in the40,000 IU q.w. group was slightly higher (70.3 kg) compared to the 150IU/kg t.i.w. group (66.8 kg), with the rest of the baseline anddemographic characteristics being very similar between the two groups.

[0377] Demographic and baseline characteristics of the 34 subjects whocompleted the study are presented in FIG. 47. There were no notabledifferences from the overall study population.

[0378] Study Completion/Withdrawal Information

[0379] Subjects were considered to have completed the study if theyparticipated for the full duration (29 days) of the study. In addition,the subject must have taken all required doses of the study drug, theymust have been compliant with the blood sampling procedures, and theymust have undergone Day 29 evaluations and procedures. The efficacypopulation included subjects receiving all required doses who completedthe study. The efficacy population was used for pharmacokinetic andpharmacodynamic data analyses. 94% of the subjects in both groupscompleted the study. One subject (1014) in the epoetin alfa 150 IU/kggroup withdrew on Day 15 of the study due to an adverse event(persistent headaches), and one subject (1006) in the epoetin alfa40,000 IU q.w. group withdrew on Day 10 (subject choice). These subjectswere not included in the efficacy population.

[0380] Analytical Results

[0381] Pharmacokinetic Results

[0382]FIG. 48 illustrates the mean serum epoetin alfa concentration-timeprofiles (uncorrected for predose endogenous erythropoietin level) forthe 150 IU/kg t.i.w. and the 40,000 IU q.w. groups during Week 4 of thestudy period. The mean serum erythropoietin concentration-time profilescorrected for predose endogenous erythropoietin level are shown in FIG.49.

[0383] The mean (SD) pre-dose endogenous erythropoietin alfaconcentrations for subjects in the 150 IU/kg t.i.w. and 40,000 IU q.w.regimen groups were 8 (0.4) and 9 (2) mIU/mL, respectively. During Week4 of the 150 IU/kg t.i.w. dosing regimen, erythropoietin concentrationsin serum (corrected for baseline erythropoietin) ranged from peakconcentrations of 75 to 284 mIU/mL [mean (SD) C_(max)=143 (54) mIU/mL]to trough level values ranging from values below the quantificationlimit of the analytical method (7.8 mIU/mL) to 40 IU/mL [mean (SD)trough concen-tration (C_(min))=18 (9) mIU/mL]. During Week 4 of the40,000 IU q.w. dosing regimen, serum erythropoietin concentrations(corrected for baseline erythropoietin) reached peak concentrations[mean (SD) C_(max)=861 (445) mIU/mL] at times ranging from 1 to 24 hours[median t_(max)=15 (range, 1-24) hours], then declinedmulti-exponentially to trough level values ranging from values below thequantification limit of the analytical method (7.8 mIU/mL) to 5.9 mIU/mL[mean (SD) trough concentration on Day 29=2.0 (1.5) mIU/mL) at the endof the dosing week on Day 29. Mean (SD) C_(min) of the 40,000 IU q.w.during the four week study period was 3.8 (4.3) mIU/mL. The terminalphase of the two dosing regimens seemed to be in parallel with mean (SD)half-life values of 19.4 (8.1) hours (n=9) and 15.0 (6.1) hours (n=9)for the 150 IU/kg t.i.w. and the 40,000 IU q.w. dosing regimens,respectively.

[0384] Mean (SD) [% CV] pharmacokinetic parameter values are presentedin FIG. 50. Bioavailability of epoetin alfa after the 40,000 IU q.w.dosing regimen relative to after the 150 IU/kg t.i.w. dosing regimen was239%.

[0385] Pharmacodynamic Results

[0386] The mean changes from baseline in percent reticulocytes,hemoglobin concentrations, and red blood cell values are summarized bytreatment group and study day in FIGS. 51, 53, and 55, respectively.Mean change from baseline for pharmacodynamic results is presented onlyfor subjects who completed the study.

[0387] Linear plots of mean change from baseline versus study day forpercent reticulocytes, hemoglobin concentrations, and red blood cellvalues are presented in FIGS. 52, 54, and 56, respectively.

[0388] Percent Reticulocytes

[0389] In both groups, mean change in percent reticulocytes increasedthrough Day 10 and gradually declined through Day 29 (FIGS. 51 and 52).

[0390] Hemoglobin

[0391] Mean hemoglobin at baseline was equivalent in the two dosagegroups, 13.4 g/dL in the 150 IU/kg t.i.w. group and 13.5 g/dL in the40,000 IU q.w. group. In both treatment groups, mean change frombaseline for hemoglobin values increased through Day 26 (FIGS. 53 and54). Mean change from baseline in hemoglobin for the 40,000 IU q.w.group mirrored the change in the epoetin alfa 150 IU/kg t.i.w. group.Overall, both groups exhibited a 3.1 g/dL increase from baseline throughDay 29.

[0392] Red Blood Cells

[0393] The mean change from baseline in red blood cell values isillustrated in FIGS. 55 and 56. In both treatment groups, mean changefrom baseline for red blood cell values increased through Day 24. Meanchange from baseline in red blood cell values for the 40,000 IU q.w.group mirrored the change in the epoetin alfa 150 IU/kg t.i.w. group.Overall, both groups exhibited a 1.0×10¹²/L increase from baselinethrough Day 29.

[0394] Pharmacokinetic/Pharmacodynamic Results

[0395] Mean pharmacodynamic parameter values (corrected for baselinevalue) are presented in FIG. 57. The dynamic responses of the two dosingregimens were similar despite the fact that serum erythropoietin AUC forthe 40,000 IU q.w. dosing regimen was larger than that for the 150 IU/kgt.i.w. dosing regimen. There were no statistically significantdifferences (p>0.05) in the AUC of % reticulocytes, AUC of hemoglobin,and the AUC of red blood cells between the two dosing regimens. Therewere no statistically significant differences (p>0.05) in the AUC ofpercent reticulocytes between male and female subjects. However, the AUCof hemoglobin and the AUC of red blood cells were statistically (p=0.038and 0.042, respectively) larger in females than in males. Thesedifferences were not clinically significant.

[0396] Safety Results

[0397] Extent of Exposure

[0398] Thirty-four (94%) of the subjects participating in the studyreceived all doses of study drug (either epoetin alfa 150 IU/kg t.i.w.[12 doses] or epoetin alfa 40,000 IU q.w. [four doses]). One subject(1014) randomized to the epoetin alfa 150 IU/kg t.i.w. group withdrewfrom the study due to an adverse event (persistent headaches) afterreceiving six doses of study drug. In the 40,000 IU q.w. group, onesubject (1006) chose to withdraw after receiving two doses of studydrug.

[0399] With one exception, all subjects received daily oral ironsupplementation as per protocol; subject 2008 (40,000 IU q.w.)discontinued oral iron supplementation on Day 19 of the study. With oneexception, all women continued with the method of birth controlpracticed prestudy (as per protocol); subject 1010 (epoetin alfa 40,000IU q.w.) discontinued oral birth control eleven days before studyinitiation.

[0400] Adverse Events

[0401] All treatment-emergent adverse events are summarized in FIG. 58.Overall, 13 (72%) of 18 subjects administered epoetin alfa 150 IU/kgt.i.w. had an adverse event compared with 12 (67%) of 18 subjectsadministered epoetin alfa 40,000 IU q.w. The majority oftreatment-emergent adverse events were mild in severity with minorqualitative differences between the two groups.

[0402] The most frequently reported adverse events were pain (22% 150IU/kg, 28% 40,000 IU q.w.), headache (28% both groups), and erythematousrash. Five (28%) of 18 subjects receiving epoetin alfa 150 IU/kg t.i.w.exhibited an erythematous rash at the cannula site in the forearm,compared with two (11%) of 18 subjects receiving epoetin alfa 40,000 IUq.w. All events were assessed by the investigator to be unrelated toepoetin alfa therapy.

[0403] Summary of All Adverse Events

[0404] Deaths, Other Serious Adverse Events, and Other SignificantAdverse Events

[0405] There were no deaths or serious adverse events during the courseof the study. One subject (1014) administered epoetin alfa 150 IU/kgt.i.w. discontinued the study due to an adverse event (persistentheadaches) on Day 15 of the study. The headaches were assessed by theinvestigator as very likely to be related to epoetin alfaadministration.

[0406] Subject 2007 (150 IU/kg t.i.w., a 28-year-old white male, had ahemoglobin level of 18.0 g/dL on Day 26. Repeat hemoglobin evaluationlater that day revealed a level of 17.6 g/dL. On Day 29, the subject'shemoglobin level was 18.2 g/dL; repeat evaluation on Day 31 revealed ahemoglobin level of 18.4 g/dL. The subject was then phlebotomized asspecified in the protocol; 450 mL of blood were removed. The subjectcompleted the study on Day 29 and hemoglobin levels were subsequentlymonitored for safety; evaluations on Days 32 and 39 revealed hemoglobinlevels of 17.7 g/dL and 16.9 g/dL, respectively.

[0407] Concomitant medications used during the study for the treatmentof adverse events included: paracetamol for headache (subjects 1014,2004 [150 IU/kg] and 1011, 2008 [40,000 IU]), tooth pain (subject 2004[150 IU/kg]), neck pain (subject 2010 [150 IU/kg]), period pain (subject1013 [150 IU/kg]), gastric flu (subject 1011 [40,000 IU]), cold (subject1013 [150 IU/kg]), and pain at cannulae sites (subject 1008 [150IU/kg]); and normal saline and chloramphenicol (subject 2004 [150IU/kg]) for ocular inflammation.

[0408] Clinical Laboratory Evaluation

[0409] Laboratory Values Over Time

[0410] The mean changes from baseline in iron, ferritin, and transferrinsaturation are summarized in FIG. 59. There were no consistent patternsin iron values or transferrin saturation that would indicate that eithertreatment resulted in clinically significant abnormalities.

[0411] The patterns reflected in ferritin changes, which were similarbetween groups, reflect expected use of iron stores for the productionof hemoglobin (FIG. 60). The fluctuating serum iron levels in bothgroups were not considered clinically meaningful.

[0412] As FIG. 60 illustrates, mean change from baseline in ferritinvalues decreased through Day 8 and remained low through Day 29,indicating continued erythropoiesis through this period. There was nonotable difference between the two groups in mean change from baselineferritin values.

[0413] Individual Subject Changes

[0414] There were no individual subject changes recorded as an adverseevent during the course of the study.

[0415] Other Safety Observations

[0416] Vital Signs

[0417] A summary of the mean changes from baseline in vital signmeasurements by study day for individual subject data are presented inFIG. 61. There were no clinically significant changes in mean vital signmeasurements for either treatment group and no significant differencesbetween groups.

[0418] Two subjects administered epoetin alfa 150 IU/kg t.i.w. exhibitedsystolic blood pressure values at or above the upper limit of 140 mmHg;none of these events were considered by the investigator to beclinically significant, and none were recorded as adverse events.

[0419] Physical Findings

[0420] There were no clinically significant changes from baseline inphysical examinations.

[0421] Safety Conclusions

[0422] Epoetin alfa administered t.i.w. at 150 IU/kg or q.w. at 40,000IU was safe and well tolerated by healthy subjects in this study. Therewere no clinically important treatment-emergent adverse events in eithertreatment group. The majority of treatment-emergent adverse events weremild in severity with minor qualitative differences between the twogroups. No subject died during the study, and there were no seriousadverse events reported. One subject administered epoetin alfa 150 IU/kgt.i.w. was phlebotomized on Day 31 of the study, due to high hemoglobinlevels. Subsequent monitoring of hemoglobin levels in this subjectrevealed no further elevation in hemoglobin levels. One subjectreceiving epoetin alfa 150 IU/kg t.i.w. withdrew from the study due toan adverse event (persistent headaches). There were no clinicallysignificant changes noted in clinical laboratory test values, mean vitalsign measurements, or physical examinations for either group; there werealso no apparent differences in the results between the two groups.

[0423] Summary and Discussion

[0424] Following administration of epoetin alfa 40,000 IU q.w., C_(max)values were six times and AUC₍₀₋₁₆₈₎ values were three times that of the150 IU/kg t.i.w. dosing regimen. Clearance after the 150 IU/kg t.i.w.dosing regimen was higher than that after the 40,000 IU q.w. dosingregimen. The time profiles of changes in percent reticulocytes,hemoglobin, and total red blood cells over the one month study periodwere similar between the two dosing regimens despite the differences inexposure of epoetin alfa in serum [in terms of AUC₍₀₋₁₆₈₎]. In addition,there were no statistically significant differences (p>0.05) in AUC ofpercent reticulocytes, AUC of hemoglobin, and total red blood cell overthe one month study period between the two dosing regimens. Although thedifferences in AUC of hemoglobin and AUC of total red blood cellsbetween male and female subjects were statistically significant, thesedifferences were not considered clinically meaningful. The data of thisstudy clearly indicate that the hemoglobin responses after the 150 IU/kgt.i.w. and the 40,000 IU q.w. dosing regimens were similar.

[0425] There was an expected difference in total exposure of epoetinalfa in serum after the 150 IU/kg t.i.w. and the 40,000 IU q.w. dosingregimens. Hemoglobin responses were similar, suggesting that the twodosing regimens can be used interchangeably.

Example 3 Comparison of PK/PD Parameters After Administration of EPREX®and PROLEASE®

[0426]FIG. 62 is a schematic representation of the model forerythropoiesis stimulating effects of rHuEpo. This model was used toestimate the kinetic and dynamic parameters for rHuEpo responses afteradministration of 8 single doses of EPREX® as well as the kineticparameters after single dose PROLEASE® administration.

[0427] Pharmacokinetics

[0428] The pharmacokinetics of 600 IU/kg/wk EPREX® administered for 4weeks was simulated using parameters obtained from the simultaneousfitting of the eight single doses. Only the Tau and Fr values wereestimated. For the INT-57 cancer regimen of 150 IU/kg/t.i.w, the F, Tau,and Vd values were fixed as indicated in FIG. 63 based on previousestimations from the earlier study (EPO-358/359). The pharmacokineticsfor single dose PROLEASE® (2400 IU/kg) were estimated and theseparameters were used to simulate the multiple dose regimen of 1800IU/kg/month. The same sets of kinetic parameters were used tocharacterize profiles for both males and females, since preliminary runsdid not show significant differences in the estimated parameters basedon gender.

[0429] Pharmacodynamics

[0430] The kinetics was fixed and used as a forcing function for drivingthe dynamic responses. Predose values were fixed as baseline levels forreticulocytes and the mean of the 48 and 96-hour values were fixed asbaseline for RBC. Lifespan parameters obtained from single doseestimation for EPREX® were fixed for further multiple dose fittings. TheSmax and SC₅₀ were estimated for the reticulocyte response after themultiple dose 600 IU/kg/wk surgery regimen. As reported in FIG. 64,these parameters do not seem to change very appreciably considering thevariability in the responses. The difference may simply reflect the factthat this was a different Phase I study from the single dose study,conducted on a new set of volunteers. Moreover, these set of parametersseem to well characterize the 150 IU/kg/t.i.w. cancer regimen too basedon the simulations shown in FIGS. 65 and 66. The reticulocyte data formales and females were analyzed separately since the estimated Smax andSC50 from the data on male subjects did not describe the responses forfemales well enough. As seen from FIG. 68, these parameters weredifferent when estimated separately for females, though it would be hardto judge whether the difference is appreciable. These parameters mayreflect some slight pharmacodynamic differences based on gender sinceall the data was obtained from a single study which might be expected tohave lower variability.

[0431] The estimated EPREX® parameters were used to simulate theresponses for both the single and double dose PROLEASE® regimens.Simulations using the models of the present invention were performed formales and females separately according to the parameters estimated forthe respective genders from EPREX® estimations. Further, the RBCresponses were simulated based on the parameters generated using thereticulocyte data, and the erythrocyte response seems to becharacterized well in all the cases for both EPREX® and PROLEASE®.

[0432] Based on the simulations as shown in FIGS. 66 and 67, it can beseen that the same set of dynamic parameters can well describe responsesto both the formulations Hence, it can be concluded that EPREX® andPROLEASE® seem to be pharmacodynamically equivalent. The models of thepresent invention predict that the differences in the responses betweenthe two formulations may be accounted for completely by the alteredkinetics. Indeed, the models of the present invention may be used tocompare the PK/PD characteristics of new forms and versions of EPO andEPO-like compounds with those currently available to provide the patientwith the most beneficial treatment regimen.

Example 4 Comparison of Different Dosing Regimens of rHuEPO

[0433] The standard dosage regimens for chronic administration of rHuEPOare 150 IU/kg/t.i.w. and 600 IU/kg/week. There may be savings in costand added convenience if patient therapy involved less frequent dosing.Therefore, the differences in hemoglobin responses for various dosageregimens of rHuEPO were examined using the models of the presentinvention to characterize and predict responses to rHuEPO which offerthe most efficient treatment regimens.

[0434] The production/loss pharmacodynamic model along with the dualabsorption pharmacokinetic model of the present invention was used.Parameters obtained from fittings of rHuEPO dynamics in healthyvolunteers (FIG. 3 and FIG. 13) were used for simulations of thedifferent dosage regimens. A baseline EPO concentration of 40 IU/L wasfixed for the simulations in all cases. The ADAPT II program was usedfor all simulations.

[0435]FIG. 68 shows the simulated hemoglobin response versus timeprofiles for several different doses and dosing regimens of rHuEPO. Allregimens produce a continual rise in Hb concentrations untilsteady-state is reached around 126 days (3024 hr). The dose of 600IU/kg/wk seems to produce the maximum increase in Hb levels. This doseand regimen can keep the rHuEPO concentrations above the threshold of 23IU/L for most periods of time causing continual increases in cell countsultimately producing higher steady-state levels of Hb. The same totaldose of 1200 IU/kg given every 2 weeks produces responses which are muchlower because the rHuEPO concentrations fall below threshold before mostof the reticulocytes are converted to erythrocytes. Moreover, thesucceeding doses of rHuEPO are also not given soon enough to elevate theconcentrations above the threshold as would occur with every weekdosing.

[0436] A similar argument can be made in comparing the 450 IU/kg/wkregimen to the 900 IU/kg every 2 weeks dosing. The 150 IU/kg t.i.w.dosing is equivalent to the 450 IU/kg/wk dosing regimen in terms of thetotal dose delivered but the dynamic profiles after thrice a week dosingyield a slightly better Hb response. As expected, the 900 IU/kg/10 daysdosing schedule yields a steady-state response profile (56% increase)better than the 1200 IU/kg/2 wk (48% increase) but lower than the 600IU/kg/wk (71% increase) regimen.

[0437] When treatment is continued for a substantial length of time, thetrue steady-state responses attained appear to differ with the variousdosage and regimens. These differences in steady state response,however, are not as apparent with the short term treatment regimen,which causes only slight elevations in Hb levels (e.g., an increase by 1unit), except for the 600 IU/kg/week treatment, which causes aconsistently higher Hb response in comparison to the other regimens. Thesimulations, using the models of the present invention, show that as thetime for readministration is decreased, greater and steadier increasesin Hb can be achieved with the same total dose. Frequent dosing helps tokeep the rHuEPO concentrations above the threshold facilitatingformation of RBC from reticulocytes. The erythrocytes, having a 40 timeslonger life-span than that of reticulocytes, persist in blood for a muchlonger time resulting in steady increases in Hb levels. Also, it is seenthat the change in response seen with more frequent administration alsodepends on the dose chosen. Switching from an every other week regimento a weekly dosing schedule affects the 600 IU/kg dose more than the 450IU/kg dose. Though better in terms of the steady-state responseachieved, thrice a week dosing may not be preferable over weekly dosingfor the same total dose because the extent to which there is improvementin response is not great enough compared to the inconvenience of morefrequent dosing. Hence, for the dosage regimens tested, a once-weekly600 IU/kg/dose of EPO is shown to provide the desired PK/PD response.

[0438] Indeed, the models of the present invention can provide anydesired dosing regimen, such as less than once daily, to less than onceweekly to less than once every two, three, or four weeks, depending onthe EPO used and the desired PK/PD response. Thus, the models of thepresent invention are not limited to use with any particular type of EPOor any specific type of dosage regimen, and can be modified and usedwith any type of EPO.

Example 5 Effects of 40,000 IU/WK Dosing of rHuEPO in Relation toPatient Body Weight

[0439] The need for repeated rHuEPO administration causes dosing basedon body weight to be inconvenient and time consuming. A switch from thispractice to dosing a definite amount irrespective of subject body weightwould facilitate clinical use of rHuEPO. Hence, simulations wereperformed with the models of the present invention in an attempt toreveal the extent of changes in expected RBC and hemoglobin responseprofiles with body weight alterations. It was intended to use thesesimulations to provide insight as to whether this change in the mode ofdosing is justifiable from a theoretical perspective.

[0440] The production/loss pharmacodynamic model along with the dualabsorption pharmacokinetic model of the present invention was used.Parameters obtained from fittings of rHuEPO dynamics in healthyvolunteers (FIG. 3 and FIG. 13) were used for simulations of thedifferent dosage regimens. A baseline EPO concentration of 40 IU/L wasfixed for the simulations in all cases. The ADAPT II program was usedfor all simulations.

[0441] The effects of subject body weight on the expected response tomaintenance therapy with rHuEPO is depicted in FIG. 69. FIG. 69 showsthe simulated RBC and Hb response versus time profiles for the regimenof 600 IU/kg/wk for 24 weeks (4032 hr) in comparison to giving a totaldose of 40000 IU/wk to subjects with body weights of 50, 70 and 90 kg.Both RBC and Hb show continual increases over the duration of rHuEPOadministration. The dose of 40000 IU/wk resembles the 600 IU/kg/wkdosing regimen assuming that most subjects have a 70 kg weight.

[0442] A change in body weight affects the volume of distribution (Vd).The rHuEPO has a Vd of 0.0558 L/kg, which is very close to plasmavolume. As body to weight increases, Vd (L) increases causing themaximum rHuEPO concentrations attained to become lower. A change in bodyweight also affects the clearance parameter Vmax (IU/hr/kg). There is anincrease in Vmax (IU/hr) with increase in body weight leading to anincrease in clearance (Vmax/(Km+C_(EPO))) of rHuEPO. As both Vmax and Vdare affected to the same extent, the elimination rate constant (i.e., kat lower concentrations) remains unchanged. In any case, body weightdifferences appear not to affect the kinetics to a significant extentbecause the terminal slope after SC administration is in fact governedby the absorption kinetics and with the SC₅₀ being very low, theterminal slope principally governs the extent of response.

[0443] The simulations show that the steady-state levels of RBC countsand Hb counts are slightly different based on body weight. The time toreach steady-state, however, is also long and therefore, the Hbresponses at early times such as 4 weeks do not differ very much (16.46,16.33 and 16.19 g/dl for 50, 70 and 90 kg subjects versus 16.35 g/dl for600 IU/kg/wk dosing).

[0444] Therefore, the data derived from the models of the presentinvention show that differences in body weight over the 50 to 90 kgrange do not contribute substantially to rHuEPO kinetics and dynamics.Therefore, dosing based on body weight might not be optimal, norimperative and a change from dosing on a body weight basis to a standardregimen of 40,000 IU/wk irrespective of body weight, is reasonable andconvenient. The 40,000 and 650 IU doses are not meant to be absolute andcontemplate a range of values in the dose that can be administered to apatient and provide the same or similar effect. Indeed, “about” 40,000or 650 IU contemplates a range of values that provide the same orsimilar effect to a patient and contemplates ranges of, in a particularembodiment, +/−1 to 20% of the IU value.

Example 6 Assessment of rHuEPO Dynamics in Cancer Patients

[0445] Cancer patients undergoing chemotherapy are often anemic.Inadequate endogenous EPO production is believed to be one of thefactors responsible for the anemic condition in these patients, andadministration of rHuEPO at a dose of 150 IU/kg three times a week(t.i.w.) has been shown to prove beneficial in correcting the anemia.Though this regimen gives adequate responses and is most commonly used,the frequent dosing required makes it inconvenient and it might not bethe best one. Therefore, an optimal dose of rHuEPO, which can be givenon a weekly regimen to yield comparable increases in hemoglobin levelsas the current thrice a week regimen, was sought. The PK/PD models ofthe present invention using data from normal subjects was used toquantitatively compare the responses in cancer patients and explainpossible causes of differences, if any, in the dynamics. The study alsoprovides an opportunity to validate the models of the present inventionso that they could be reliably used for predictive purposes in thefuture.

[0446] The data for the cancer patients was obtained from RWJPRI. Thiswas an open-label, randomized, controlled, parallel group, multicenterstudy carried out in 150 anemic cancer patients having solid tumors andreceiving platinum-containing chemotherapy (cisplatin or carboplatin).Different dose levels of PROCRIT® (Epoetin alfa, Amgen) which includedweekly SC doses of 300, 450, 600, 900 IU/kg and an SC dose of 150 IU/kgt.i.w. were administered to 5 groups of subjects (25 patients per group)for a period of 12 weeks. The control group received no treatment.Patients were required to be 18 years of age or older, have prestudy Hb≦10 g/dl, corrected reticulocyte counts ≦3%, platelets ≧25,000cells/mm³, creatinine ≦2.0 mg/ml, negative stool occult blood, noevidence of hemolysis, and normal serum folate and Vitamin B₁₂ levels.Also, only those subjects that had not required blood transfusion onemonth prior to randomization and that were not iron-deficient wereincluded in the study. The hemoglobin count was measured as the primarypharmacodynamic end point.

[0447] The parameters obtained from fittings of the kinetic and dynamicdata for normal volunteers were used to simulate responses afteradministration of the different doses and regimens of rHuEPO to thecancer patients. The production/loss pharmacodynamic model along withthe dual absorption kinetic model of the present invention was used.Parameters obtained from fittings of the rHuEPO dynamics in healthyvolunteers (FIG. 3 and FIG. 13) along with a baseline EPO concentrationof 40 IU/L were used as a starting point for simulations of thedifferent dosage regimens, and the effects of changing variousparameters on the responses were investigated. The ADAPT II program wasused for all the simulations.

[0448] FIGS. 70A-70E show the hemoglobin data and simulations ofreticulocytes, RBC and Hb responses for anemic cancer patients who weregiven different doses and regimens of rHuEPO. As seen in the figures,the patients seem to respond favorably to therapy in general withcontinual increases in Hb concentrations. There is only a very slightresponse associated with the 300 IU/kg dosage regimen. With increases inthe weekly dose, there seems to be an increase in the extent ofresponse. The highest dose of 900 IU/kg/week, however, does not produceconsiderably higher increases in Hb levels than the 600 IU/kg/wk dose.The data show that a weekly regimen of 600 IU/kg produces responsesslightly better than the 150 IU/kg t.i.w. regimen. It can also be seenfrom the figures that the simulations using parameters from healthysubjects predict responses that are higher compared to those actuallyobserved in cancer patients. Differences in the kinetics and/or dynamicsof rHuEPO in cancer patients compared to healthy subjects can explainthe cause of these altered response profiles. Simulations were thereforeperformed altering selected parameters in the pharmacodynamic model toexplain these differences.

[0449] Erythroid hypoplasia of the bone marrow, decreased RBC survival,and decreased reticulocytosis are reported (see, e.g., Abels, 1992.Semin. Oncol. 19:29-35) to be some of the possible causes of anemia inchronic disease. Anticancer drug therapy is also thought to be one ofthe principal causes of anemia in these patients. (see, e.g., Matsumoto,et al., 1990. Br. J. Pharmacol. 75:463-68.) A reduction in the Ks valueby ⅓rd, indicating a lowered intrinsic production rate of cells and/or alower S_(max) could explain the diminished responses as seen in thesimulations. Cancer patients have baseline EPO concentrations, which arehigher than normal, but inappropriately low for the degree of anemia.(see, e.g., Case, et al., 1993. J Natl. Cancer Inst. 85:801-806 andMiller, et al., 1990. N. Engl. J. Med. 322:1689-99. The baseline EPOconcentrations are reported to range from lower than 40 to higher than500 U/L (see, e.g., Ludwig, et al., 1994. Blood 84:1056-63, Case et al.,supra, Abels, supra, and Miller et al., supra) depending on the severityand type of anemia associated with the cancer and chemotherapy. BaselineEPO levels greater than 500 IU/I have been reported to indicateunresponsiveness to rHuEPO therapy. (see, e.g., Ludwig et al., supra.)Increasing the baseline EPO concentrations in the model to 70 U/L,signifying a decreased sensitivity of the system to EPO, shifted theresponse-time profiles down and gave a better fit of the data. Sincedosing was done every week, concentrations remained well above thethreshold allowing the conversion of most reticulocytes to erythrocytes.Therefore, a change in the threshold did not significantly help inshifting the curves, which suggests that reticulocyte-RBC conversionprocess may not be significantly affected in these patients. Undernormal conditions, erythrocytes live for a period of 2880 hours andaccording to the production/loss model, the true steady-state is reachedone life-span after the beginning of production of new erythrocytes. Anyreduction in RBC life span would cause this steady state to be reachedat an earlier time and be lower in these patients. This possibility wasnot demonstrated because the dosing was not carried out long enough toallow attainment of true steady-state under normal conditions in thestudy. As mentioned earlier, another possible cause of diminishedresponses in cancer patients could be differences in thepharmacokinetics of rHuEPO. In healthy volunteers, rHuEPO undergoesflip-flop kinetics after SC administration causing concentrations tostay above baseline for prolonged periods of time allowing continuedstimulation of the production of new cells. The slow first-order inputvia lymphatics was assumed to contribute to this phenomenon and anyalterations in the physiological functioning of the lymphatics due tothe disease state and chemotherapy may abate the slow delivery leadingto lower responses.

[0450] Although the weekly regimen of 600 IU/kg requires a higher totaldose compared to 150 IU/kg t.i.w., it produces better Hb responses incancer patients and is a more convenient dosing schedule for rHuEPOmaintenance therapy. Therefore, a change from the current regimen to 600IU/kg/wk might be preferable according to the models of the presentinvention. The PK/PD model developed can account for differences inresponses due to disease conditions such as cancer, and the simulationspredict that a lower Ks value and/or higher baseline EPO levels with orwithout alterations in the pharmacokinetics of rHuEPO could beresponsible for the blunted responses seen in these patients.

[0451] With reference to the preceding detailed description and specificexamples, one skilled in the art will understand that the PK/PD modelingsystem of the present invention may be used in a variety of situations.For example, a practitioner may want to adjust the EPO dosage regimen toachieve a desired pharmacokinetic response in a patient, such as serumEPO concentration. The practitioner can use the systems of the presentinvention to accomplish this result. Alternatively, the practitioner maywant a specific pharmacodynamic response in patient, such as a specificincrease in hemoglobin levels. The practitioner can use the systems ofthe present invention to determine which EPO dosage regimen will becapable of achieving the desired result. In another aspect, thepractitioner may want to determine what type of pharmacokinetic andpharmacodynamic outcome will result from a specific EPO dosage regimen.Again, the practitioner will be able to use the PK/PD models of thepresent invention to make this determination.

Example 7 Immunogenicity of EPO in Dogs During One-Month Dosing Regimens

[0452] This study was designed to evaluate the immunogenicity of EPOformulations in immunosuppressed and non-immunosuppressed beagle dogs.Pharmacodynamics and pharmacokinetic profiles of EPO formulations wereexamined.

[0453] Regulatory Compliance

[0454] Good Laboratory Practice (GLP): This study was not conducted instrict compliance with the U.S. Food and Drug Administration's GLPRegulations for Nonclinical Laboratory Studies (21 CFR, Part 58), yetwas performed according to the protocol and applicable Oread standardoperating procedures.

[0455] Animal Care and Use: Animal studies were conducted in accordancewith the NRC “Guide for the Care and Use of Laboratory Animals”,(Revised 1996) and the USDA “Laboratory Animal Welfare Act, Aug. 24,1966, Pub.L.89-544 and subsequent amendments. Oread Met/PK is an AAALACaccredited facility.

[0456] Identification and Source

[0457] Formulations:

[0458] EPREX®, one 2000 IU/mL and one 10,000 IU/mL Saline as control

[0459] Storage

[0460] EPO formulations were stored refrigerated (˜4′.C) protected fromlight when not used on study. Unused formulations were returned toRWJPRI following dosing or destroyed. Study Animals Species: Dog Strain:Beagle Sex: Male Source: Harlan Sprague Dawley, Inc. Indianapolis,Indiana 46299 Age at Dosing: 8-9 months Target Weight at First Dosing:9-12 kg Identification Method: Tattoo applied by animal supplier Numberon Study: 18 (N = 3 dogs/group)

[0461] Housing

[0462] Dogs were group housed by treatment group within kennels in a dogholding room and acclimated to handling and sample collection prior todose administration. Quarantine were at least 5 days prior to doseadministration. At the end of the quarantine period, the health of allanimals was confirmed by study personnel. During the study/collectionperiod, the dogs remained in group housing unless necessary due tohealth conditions. Kennels were labeled with the animals' and protocolnumbers.

[0463] Environmental Conditions

[0464] Animal rooms were maintained at 23±3° C. with a relative humidityof 50±15% and a 12-hour light/dark cycle. There was at least 10 room airchanges per hour.

[0465] Diet and Water

[0466] Dogs had access to Purina Certified Canine Diet® #5007 and waterad libitum when on study. Results of food analysis (certificate ofanalysis provided by the vendor) and water analysis (dissolved solids,microbial content, selected elements, heavy metals, and chlorinatedhydrocarbons) were maintained in the raw data file. No contaminants werereasonably expected to be present in feed or water at levels sufficientto interfere with the results of the study.

[0467] Justification of Dose and Species

[0468] The dose was selected based on existing data obtained fromformulations evaluated in previous studies. This study was conducted inimmunosuppressed and nonimmunosuppressed beagle dogs to evaluate theimmunogenicity of EPO formulations, and the PK/PD profiles of two dosingregimens. The number of dogs that were used was the minimum numbernecessary to provide scientifically valid results. No acceptable invitro models were available. Purpose bred beagle dogs are routinely usedfor the conduct of pharmacokinetic, pharmacodynamic, and toxicologicalstudies to meet regulatory requirements.

[0469] Study Design

[0470] Summary

[0471] Beagle dogs (N=3 dogs/group, 6 groups) were randomly assigned totreatment groups. On Day-2, three groups of dogs were administered asingle oral dose of cyclosporin (25 mg/kg). Thereafter, the three groupsof dogs received a daily maintenance dose of cyclosporin (10 mg/kg). Twodosing regimens with EPREX® and vehicle were examined inimmunosuppressed and nonimmunosuppressed dogs. All formulations andvehicle were administered subcutaneously (sc) either daily or weekly. Atdesignated times over a four-week period, blood samples were collected.The injection site was monitored daily and body weights obtained weekly.Dogs were euthanized or donated to another research institute followingthe last collections.

[0472] Preparation and Test Formulations Dose Administration

[0473] Day 1

[0474] Test formulations were initially administered to the dogs on Day1 (see table below). All formulations were administered at the volumespecified in the following table.

[0475] Dose were drawn-up into a syringe fitted with appropriate gaugeneedle. The SC dose was administered in the dorsal region. Dose siteswere clipped prior to dosing and marked with indelible ink. EPO Immuno-Dose Volume Dose Group Treatments suppressed (IU/kg) (mL/kg) FrequencyRoute 1 EPREX ® Yes  50 25¹ Daily SC 2 EPREX ® No  50 25¹ Daily SC 3EPREX ® Yes 600 60² Weekly SC 4 EPREX ® No 600 60² Weekly SC 5 SalineYes NA 60 Weekly SC 6 Saline No NA 60 Weekly SC

[0476] Observations, Sample Collection, and Processing

[0477] Dogs and injection sites were monitored daily. Any abnormalappearance or behavior was noted and evaluated. Body weights wererecorded once weekly.

[0478] At designated primary time points (see below), approximately 2 mLof blood were collected via the jugular vein into heparinizedVacutainers® (Becton Dickinson, Franklin Lakes, N.J.). In case ofjugular vein failure, blood was collected via the cephalic vein andnoted. The primary blood collection, obtained prior to cyclosporinadministration, was used to harvest plasma. The blood was placed on ice,centrifuged (1500×g, 10 min, ˜4° C.), and plasma collected. Plasma wasfrozen at −20 C for shipment.

[0479] At the secondary blood collection, approximately 2 mL of bloodcollected using a Vacutainer® containing EDTA, was obtained in themorning and placed on ice. The secondary collection was stored at ˜4° C.as whole blood and used for reticulocyte, hemoglobin, and total redblood cell measurements.

[0480] Primary Blood Collection Time Points:

[0481] Groups 1 & 2 (daily EPREX®): Predose,1, 3, 8, 12, 16 and 24 h onDays 1 and 28, and predose on Days 3, 7, 14, 21, and 24.

[0482] Groups 3-6 (weekly EPREX® or saline): Predose, 1, 3, 8, 12, 24,48, 72, and 96 h on Days 1 and 22, and predose on Days 7 and 14.

[0483] Secondary Blood Collection Time Points:

[0484] All Groups: Predose on Days 1, 3, 7, 10, 14, 17, 21, 24 and 28.

[0485] As necessary during the study, additional serum samples werecollected to assess renal and liver function. Within 24 h of the lastsample collection, dogs were euthanized with an intravenous overdose ofbarbiturate euthanasia solution or donated to another research facility.

[0486] Sample Analysis

[0487] Collected whole blood (on EDTA) was analyzed for reticulocytes,hemoglobin, and total red blood cells.

[0488] Results:

[0489] The results from this study are presented in FIGS. 71 and 72. Theresults from this study indicate that the dynamic responses (patterns ofrise in hemoglobin and red blood cells) over the 4 week study period aresimilar after the dosing regimens of 50 IU/kg/day and 600 IU/kg/week inimmunosuppressed or non-immunosuppressed dogs.

Example 8 PK/PD Modeling of Recombinant Human EPO After Three IV and SixSC Dose Administrations in Male Cynomolgus Monkeys

[0490] Objectives:

[0491] The purpose of this study was to utilize the PK/PD model of thepresent invention to characterize the pharmacokinetics (PK) andpharmacodynamics (PD) profiles of rHuEpo in terms of increasedreticulocyte, red blood cell and hemoglobin counts in blood after IVadministration of three single doses and SC administration of six singledoses of rHuEpo (EPREX®) in male cynomolgus monkeys.

[0492] Methods:

[0493] Data were obtained from two studies performed by RWJPRI (SbiStudy Nos 0876-49 and 0875-49 and RWJPRI Study Nos DM99146 and DM99124,December 1999). One study was a parallel group study performed in 12male cynomolgus monkeys (Sbi Study No 0876-49 and RWJPRI Study No.DM99146, December 1999). Monkeys were divided into 4 groups, one groupbeing the control while the other three being injected intravenouslywith 500, 2000 and 4000 IU/kg of EPREX®. Blood samples were drawnpredose and up to 48 hours for measuring rHuEpo concentrations. Theother study was a parallel group study done in 21 male cynomolgusmonkeys which were divided into 7 groups with 3 monkeys per group SbiStudy No. 0875-49 and (RWJPRI Study No. DM99124, December 1999). Thecontrol group received subcutaneously sterile saline while the remainingsix groups were administered 400, 1000, 2400, 5000, 20000 and 40000IU/kg of EPREX® subcutaneously. Animals were assigned so as to have auniform body weight distribution across groups. Blood samples were drawnpredose and at various times after administration up to day 28 forrHuEpo concentrations as well as reticulocyte, erythrocyte andhemoglobin counts. The mean data were used for this analysis.

[0494] Model: A schematic representation of the PK/PD model is depictedin FIG. 73.

[0495] Pharmacokinetics: A 2-compartment model was chosen to account forthe polyexponentiality in the kinetic profiles upon IV administration.Non-compartmental analysis indicated non-linearity in the kinetics,which was modeled using the Michaelis-Menten disposition function. Adual absorption kinetic model with a rapid zero-order input of afraction of the dose followed by a slow first-order input of theremainder was used to characterize the absorption of rHuEpo upon SCadministration. The six single SC doses as well as the three IV doseswere fitted simultaneously to this model to obtain a common set ofparameters to characterize all the data.

[0496] The differential equations used for modeling the intravenouskinetics were:$\frac{{Ap}}{t} = {{{- V}\quad \max \quad \bullet \quad {{Ap}/\left( {{{Km}\quad \bullet \quad {Vd}} + {Ap}} \right)}} - {{k12}\quad \bullet \quad {Ap}} + {{k21}\quad \bullet \quad {At}}}$$\frac{{At}}{t} = {{{k12}\quad \bullet \quad {Ap}} - {{k21}\quad \bullet \quad {At}}}$

[0497] The SC data were modeled with the following equations:$\begin{matrix}{\frac{{Ap}}{t} = {{{ko}\left( {0 - \tau_{1}} \right)} + {k_{1}\left( {t > \tau_{1}} \right)} -}} \\{{{V\quad \max \quad \bullet \quad {{Ap}/\left( {{{Km}\quad \bullet \quad {Vd}} + {Ap}} \right)}} - {{k12}\quad \bullet \quad {Ap}} + {{k21}\quad \bullet \quad {At}}}}\end{matrix}$$\frac{{At}}{t} = {{{k12}\quad \bullet \quad {Ap}} - {{k21}\quad \bullet \quad {At}}}$where ko = 0  when  t > τ₁${ko} = {{\frac{F\quad \bullet \quad \left( {1 - {Fr}} \right)\quad \bullet \quad {Dose}}{\tau_{1}}\quad {when}\quad 0} < t \leq \tau_{1}}$k₁ = 0  when  t ≤ τ₁ andk₁ = ka   •   F   •   Fr   •   Dose   •   ^(−(ka   •   (t − τ₁))  )  when  t > τ₁

[0498] Ap represents the amount of drug in the plasma while Atrepresents the drug in the peripheral compartment (i.e, tissue). Themicroconstants k12 and k21 are first order rates of transfer between thecentral (plasma) and peripheral compartments. Vmax and Km are theMichaelis-Menten constants representing the capacity of the process andconcentration at which one-half Vmax are reached. The Fr is the fractionof the dose associated with the first-order pathway of absorption k1.The time period (tau1) for the zero-order input (k0) was fixed to 10hours based on the data and initial runs. A single first-order rate ofabsorption ka could describe all the doses except the lowest dose (400IU/kg) for which a separate ka was estimated. The bioavailability Fappeared to change with dose and was estimated for the lowest two dosesand fixed to 100% for the remaining doses.

[0499] The catenary aging pharmacodynamic model (FIG. 73) with twoprecursor cell compartments having different lifespans was used formodeling the pharmacodynamics of EPREX®. Stimulation of production wasassumed to occur at the production rates of both precursor compartments.The k0 represents the zero order production rate of cells while T_(R)and T_(RBC) stand for the lifespans of the reticulocyte and red bloodcells.

[0500] The baseline rHuEpo concentrations were assumed to be zero andhence the baseline reticulocyte level was given by k0 T_(R). Thedifferential equation used for estimation purposes were as follows:$\begin{matrix}{\frac{R}{t} = {{k0}\quad {\bullet\left( {{\left( {1 + {S\left( {t - {TP2}} \right)}} \right)\quad \bullet \left( {1 + {S\left( {t - {TP1} - {TP2}} \right)}} \right)} -} \right.}}} \\\left. {\left( {1 + {S\left( {t - {TP2} - T_{R}} \right)}} \right)\quad {\bullet \left( {1 + {S\left( {t - {TP1} - {TP2} - T_{R}} \right)}} \right)}} \right)\end{matrix}$

[0501] where the stimulation function is given by the Hill equation,with gamma fixed to 1.${S(t)} = \frac{S\quad {\max \cdot \left( C_{EPO} \right)^{\gamma}}}{{SC}_{50}^{\gamma} + \left( C_{EPO} \right)^{\gamma}}$

[0502] The reticulocyte numbers after administration of the six doselevels of EPREX® were fitted to the above equation to get a single setof dynamic parameters characterizing the data across all the doses. Theparameters for the kinetic model were fixed and used as the forcingfunction for the dynamics. The predose reticulocyte counts were fixed tobe the steady-state baseline values.

[0503] The dynamic parameters obtained from the reticulocyte fittingswere used to simulate the RBC numbers and hemoglobin levels for all thedoses. The 48 hour RBC count was used as the baseline while thehemoglobin content per cell was fixed for each group from the ratio ofthe predose hemoglobin count to the predose total number of cells(RBC+reticulocytes) for that group.

[0504] The differential equations used for simulation purposes were asfollows: $\frac{{RBC}}{t} = {{k0} \cdot \begin{pmatrix}{{\left( {1 + {S\left( {t - {TP2} - T_{R}} \right)}} \right) \cdot \left( {1 + {S\left( {t - {TP1} - {TP2} - T_{R}} \right)}} \right)} -} \\{\left( {1 + {S\left( {t - {TP2} - T_{R} - T_{RBC}} \right)}} \right) \cdot \left( {1 + {S\left( {t - {TP1} - {TP2} - T_{R} - T_{RBC}} \right)}} \right)}\end{pmatrix}}$

 Hb _(t) =Hb _(cell)·(RBC _(t) +R _(t))

[0505] Results:

[0506]FIG. 74 shows the fittings for the rHuEpo concentration-timeprofiles after administrations of three single intravenous doses and sixsingle SC doses of EPREX®. The parameters obtained are listed in FIG.75. The two compartment kinetic model with non-linear disposition couldadequately capture the multiphasic IV kinetic profiles although theterminal phase for the lowest dose seems to be slightly overestimated. Ahigh Km value was estimated which indicates that the non-linearity indisposition is mild and would be prominent only at high doses. Thecentral volume of distribution Vd was estimated to 57 ml/kg, which isclose to the blood volume. For the SC administrations, thebioavailability substantially increased with dose with the lowest doseshowing a bioavailability of 26.8% and the next higher dose 73%. Thelowest dose has a slightly different ka value compared to the rest ofthe doses. It can be inferred from the parameter estimates that a majorfraction of the bioavailable dose follows the slow first-order pathway.The zero-order route of entry seems to be fast and accounts for asmaller fraction (35.5%) of the bioavailable dose.

[0507] The reticulocyte fittings are shown in FIGS. 76a, 76 b, and 77lists the pharmacodynamic parameters estimated. The lag time which isaccounted for by the second precursor compartment TP2 was small (˜15hr). The estimated reticulocyte lifespan was close to 6 days. The Smax,which signifies the maximum possible increase in production rate, was3.133 whereas a high SC₅₀ value of 842.5 IU/L was estimated. FIGS. 78aand 78 b show the simulations for the RBC numbers and FIGS. 79a and 79 bare simulations for the hemoglobin response.

[0508]FIG. 80 shows the pharmacodynamic parameters obtained afteradministration of EPREX® in healthy humans. The pharmacokinetic modelwas simplified from a 2-compartment to a 1-compartment model based onthe IV concentration-time profiles. The pharmacodynamic model wasextended by employing control of cell production by endogenous EPOlevels and an extra component accounting for the body's natural feedbackmechanism was also included (FIG. 81). This was done by assuming thatreticulocytes cause a feedback inhibition of their own production byreducing the production rate of the earliest cells represented by the P1compartment. It was also assumed that it takes a time of TP0 hours forthis inhibition to take effect. The inhibition was modeled using theHill function. A comparison of the pharmacodynamic parameter estimatesbetween monkeys and humans shows that the compartment lifespanparameters are very similar between these species. The Smax and the SC50values seem to differ between species, but this might be due to theadded complexity of counter-regulation and baseline Epo concentrationsin the model for rHuEpo effects on reticulocytes in human.

[0509] Discussion:

[0510] Pharmacokinetics: Upon IV administration, the kinetics followed abiexponential decline, which was captured using a two-compartment modelwith non-linear disposition. The primary site of action of rHuEpo is thebone marrow, which is a highly perfused tissue, and so the peripheralcompartment in the model may only represent some non-specific binding ofrHuEpo. The terminal phase for the lowest dose was overestimated whichcould be due to unavailability of concentration measurements for thelater time points or possibly a different kinetic behavior in the rangeof this dose. Similar to humans, the estimated Vd was very close to theblood volume in monkeys, and the kinetics were mildly non-linear asindicated by the high Km value.

[0511] After SC administration, the peak concentrations of rHuEpo wereattained within one day and rHuEpo remained in circulation for a muchlonger time compared to that after IV administration due to theoccurrence of the flip-flop phenomenon with ka governing the terminalphase. These data patterns were acceptably captured using a multiphaseabsorption model. The initial concentrations for the highest SC dosewere slightly overestimated. However, this should be acceptableconsidering the fact that a single set of parameters was used todescribe all the dose levels. Our dual absorption kinetic model can beused to explain the different pathways for absorption of the drug fromthe SC site. The rapid zero-order input of a part of the dose may beexplained by a direct entry via blood vessels in the subcutaneous siteto the blood. On the other hand, another fraction of the dose can beassumed to enter the lymphatics and undergo a slow process offirst-order absorption from the lymph to the blood. This would alsoexplain the 10-hour time lag for start of the first order absorption.The bioavailability increased with dose and was 100% for doses of 2400IU/kg and higher. This same dual absorption model well characterized theSC kinetics in human and showed a similar trend of increasingbioavailability with dose. However, unlike in monkeys, the modelpredicted a major fraction of the dose to be absorbed via the zero-orderpathway in humans.

[0512] Pharmacodynamics: The reticulocyte counts started rising within48 hours and peaked around 10 days after which they started dropping andreturned to baseline levels by 20 days. Our catenary-aging model seemsto characterize the data well. Simulations in FIGS. 78a, 78 b, 79 a and79 b show that the reticulocyte dynamics can be readily used to predictthe change in RBC numbers as well as hemoglobin counts.

[0513] The exact mode of action of erythropoietin is still not fullyunderstood. The primary action of rHuEpo was thought to be stimulationof the proliferation of early progenitor cells. However, there isevidence from studies on experimental animals that erythropoietin actson the differentiated erythroblasts as well (Krantz et al.,Erythropoietin and the regulation of erythropoesis, 1970, The Universityof Chicago Press). This school of thought has led to the proposal thatrHuEpo acts on the mature erythroblasts to give rise to an early 24-hourreticulocyte response followed by a macrocytosis due to an additionaleffect on normoblasts. Based on this theory, we developed themechanistic catenary-aging model with rHuEpo stimulation occurring attwo precursor cell populations, which might represent the erythroblastsand the earlier progenitor cells.

[0514] Erythroblasts are known to undergo 2 to 5 cell divisions with amean maturation or turnover time of 11 to 48 hours depending on thespecies (Id., Aplen et al., 1959, Ann. N.Y. Acad. Sci., 77:753, Osgood,E., 1954, Blood, 9:1141, and Fliedner et al., 1959, Acta. Haematol.,22:65). Our model predicts that there is an 15 hr lag time before thenewly produced reticulocytes are actually released into circulation, andthis reflects the erythroblast maturation time.

[0515] The estimated reticulocyte lifespan is 6 days. In humans, thenormal lifespan of cells in the reticulocyte stage is around 3.5 days inthe marrow and 1 to 2 days in the blood (Hillman et al., 1967, Sem.Haematol., 4(4): 327). However, in animal models of severe anemia, ithas been demonstrated that the marrow reticulocyte pool is shifted tothe circulation (Id., and Bessis et al., 1973, Living blood cells andtheir ultrastructure, Verlag New York-Heidelberg-Berlin). Thesedisplaced marrow reticulocytes take up to 3 days longer than normalreticulocytes to produce erythrocytes. Hence we could expect that theaverage lifespan of reticulocytes estimated by our model reflects thesum of the maturation times in the marrow and blood.

[0516] It has been reported in literature that in humans, it takes anaverage of 5 days for an erythroid precursor to form a reticulocyte inthe marrow (Krantz et al., supra). This time actually reflects the sumof the times a cell spends in the P1 and P2 compartments, which wasestimated to be 85 hours.

[0517] In conclusion, the rHuEPO kinetics and dynamics seems to befairly similar across species (monkey and human) and the models of thepresent invention can well approximate the kinetics and dynamics ofrHuEpo effects and gives realistic estimates of the cell aging timeparameters.

[0518] Various modifications and variations of the described examplesand systems of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in related fields areintended to be within the scope of the following claims.

We claim:
 1. A method for obtaining optimized EPO dosage regimens for adesired pharmacodynamic response in a patient comprising the steps of:(a) choosing one or more EPO dosage regimens; (b) using apharmacokinetic/pharmacodynamic model to determine the pharmacodynamicprofile of said one or more EPO dosage regimens; and (c) selecting saidone or more EPO dosage regimens that provide said desiredpharmacodynamic response based on said pharmacodynamic profile.
 2. Themethod of claim 1, wherein said pharmacodynamic response comprises ofone or more of the group consisting of reticulocyte number, RBC number,and hemoglobin level.
 3. The method of claim 1, wherein said patient isanemic.
 4. The method of claim 3, wherein said anemia comprises EPOconcentration related anemia.
 5. The method of claim 4, wherein saidanemia comprises end-stage renal or renal failure related anemia.
 6. Themethod of claim 4, wherein said anemia comprises cancer chemotherapyrelated anemia.
 7. The method of claim 4, wherein said anemia comprisesAIDS drug therapy related anemia.
 8. The method of claim 4, wherein saidanemia comprises drug related anemia.
 9. The method of claim 8, whereinsaid drug include cisplatin and zidovudine.
 10. The method of claim 1,wherein said patient is undergoing autologous transfusion prior tosurgery.
 11. The method of claim 1, wherein said patient is recoveringfrom allogenic bone marrow transplant.
 12. The method of claim 1,wherein said patient is afflicted with rheumatoid arthritis.
 13. Themethod of claim 1, wherein said dosage regimens are subcutaneous dosageregimens.
 14. A method for obtaining optimized EPO dosage regimens for adesired pharmacodynamic response in a patient comprising the steps of:(a) selecting one or more desired pharmacodynamic responses; (b) using apharmacokinetic/pharmacodynamic model to determine EPO dosage regimensthat provides said desired one or more pharmacodynamic responses; and(c) selecting the one or more EPO dosage regimens that provide saiddesired pharmacodynamic responses.
 15. The method of claim 14, whereinsaid pharmacodynamic response comprises of one or more of the groupconsisting of reticulocyte number, RBC number, and hemoglobin level. 16.The method of claim 14, wherein said patient is anemic.
 17. The methodof claim 16, wherein said anemia comprises EPO concentration relatedanemia.
 18. The method of claim 17, wherein said anemia comprisesend-stage renal or renal failure related anemia.
 19. The method of claim17, wherein said anemia comprises cancer chemotherapy related anemia.20. The method of claim 17, wherein said anemia comprises AIDS drugtherapy related anemia.
 21. The method of claim 17, wherein said anemiacomprises drug related anemia.
 22. The method of claim 21, wherein saiddrug include cisplatin and zidovudine.
 23. The method of claim 14,wherein said patient is undergoing autologous transfusion prior tosurgery.
 24. The method of claim 14, wherein said patient is recoveringfrom allogenic bone marrow transplant.
 25. The method of claim 14,wherein said patient is afflicted with rheumatoid arthritis.
 26. Themethod of claim 14, wherein said dosage regimens are subcutaneous dosageregimens.
 27. A system for selecting an optimal EPO dosage regimens fora patient using a pharmacokinetic/pharmacodynamic model comprising: (a)a processor that is controlled in accordance with a set of programinstructions that determine the steps implemented by saidpharmacokinetic/pharmacodynamic model; (b) a memory coupled to saidprocessor, said memory storing the set of program instructions andparameters used by said pharmacokinetic/pharmacodynamic model; and (c) auser interface, coupled to said processor, said user interface enablinga user to input parameters used by said pharmacokinetic/pharmacodynamicmodel.
 28. The method of claim 27, wherein said pharmacodynamic responsecomprises of one or more of the group consisting of reticulocyte number,RBC number, and hemoglobin level.
 29. The method of claim 27, whereinsaid patient is anemic.
 30. The method of claim 29, wherein said anemiacomprises EPO concentration related anemia.
 31. The method of claim 30,wherein said anemia comprises end-stage renal or renal failure relatedanemia.
 32. The method of claim 30, wherein said anemia comprises cancerchemotherapy related anemia.
 33. The method of claim 30, wherein saidanemia comprises AIDS drug therapy related anemia.
 34. The method ofclaim 30, wherein said anemia comprises drug related anemia.
 35. Themethod of claim 34, wherein said drug include cisplatin and zidovudine.36. The method of claim 27, wherein said patient is undergoingautologous transfusion prior to surgery.
 37. The method of claim 27,wherein said patient is recovering from allogenic bone marrowtransplant.
 38. The method of claim 27, wherein said patient isafflicted with rheumatoid arthritis.
 39. The method of claim 27, whereinsaid dosage regimens are subcutaneous dosage regimens.
 40. A computerprogram for obtaining optimized EPO dosage regimens for a desiredpharmacodynamic response in a patient comprising: (a) computer code thatdescribes a pharmacokinetic/pharmacodynamic model for EPO, said codeproviding for selection of one or more desired pharmacodynamic responsesand the use of said pharmacokinetic/pharmacodynamic model to determineone or more EPO dosage regimens that provide said desired one or morepharmacodynamic responses; and (b) computer readable medium that storessaid computer code.
 41. The method of claim 40, wherein saidpharmacodynamic response comprises of one or more of the groupconsisting of reticulocyte number, RBC number, and hemoglobin level. 42.The method of claim 40, wherein said patient is anemic.
 43. The methodof claim 42, wherein said anemia comprises EPO concentration relatedanemia.
 44. The method of claim 43, wherein said anemia comprisesend-stage renal or renal failure related anemia.
 45. The method of claim43, wherein said anemia comprises cancer chemotherapy related anemia.46. The method of claim 43, wherein said anemia comprises AIDS drugtherapy related anemia.
 47. The method of claim 43, wherein said anemiacomprises drug related anemia.
 48. The method of claim 47, wherein saiddrug include cisplatin and zidovudine.
 49. The method of claim 40,wherein said patient is undergoing autologous transfusion prior tosurgery.
 50. The method of claim 40, wherein said patient is recoveringfrom allogenic bone marrow transplant.
 51. The method of claim 40,wherein said patient is afflicted with rheumatoid arthritis.
 52. Themethod of claim 40, wherein said dosage regimens are subcutaneous dosageregimens.
 53. A computer program for obtaining optimized EPO dosageregimens for a desired pharmacodynamic response in a patient comprising:(a) computer code that describes a pharmacokinetic/pharmacodynamic modelfor EPO, said code providing for user selection of one or more EPOdosage regimens and the use of said pharmacokinetic/pharmacodynamicmodel to determine a pharmacodynamic response for said one or morerHuEPO dosage regimens; and (b) computer readable medium that storessaid computer code.
 54. The method of claim 53, wherein saidpharmacodynamic response comprises of one or more of the groupconsisting of reticulocyte number, RBC number, and hemoglobin level. 55.The method of claim 53, wherein said patient is anemic.
 56. The methodof claim 55, wherein said anemia comprises EPO concentration relatedanemia.
 57. The method of claim 56, wherein said anemia comprisesend-stage renal or renal failure related anemia.
 58. The method of claim56, wherein said anemia comprises cancer chemotherapy related anemia.59. The method of claim 56, wherein said anemia comprises AIDS drugtherapy related anemia.
 60. The method of claim 56, wherein said anemiacomprises drug related anemia.
 61. The method of claim 60, wherein saiddrug include cisplatin and zidovudine.
 62. The method of claim 53,wherein said patient is undergoing autologous transfusion prior tosurgery.
 63. The method of claim 53, wherein said patient is recoveringfrom allogenic bone marrow transplant.
 64. The method of claim 53,wherein said patient is afflicted with rheumatoid arthritis.
 65. Themethod of claim 53, wherein said dosage regimens are subcutaneous dosageregimens.
 66. A method for obtaining optimized EPO dosage regimens for adesired pharmacokinetic response in a patient comprising the steps of:(a) choosing one or more EPO dosage regimens; (b) using apharmacokinetic/pharmacodynamic model to determine the pharmacokineticprofile of said one or more EPO dosage regimens; and (c) selecting theone or more EPO dosage regimens that provide said desiredpharmacokinetic response based on said pharmacokinetic profile.
 67. Themethod of claim 66, wherein said pharmacokinetic response comprises ofone or more of the group consisting of serum EPO levels, bioavailablity,and threshold level.
 68. The method of claim 66, wherein said patient isanemic.
 69. The method of claim 68, wherein said anemia comprises EPOconcentration related anemia.
 70. The method of claim 69, wherein saidanemia comprises end-stage renal or renal failure related anemia. 71.The method of claim 69, wherein said anemia comprises cancerchemotherapy related anemia.
 72. The method of claim 69, wherein saidanemia comprises AIDS drug therapy related anemia.
 73. The method ofclaim 69, wherein said anemia comprises drug related anemia.
 74. Themethod of claim 73, wherein said drug include cisplatin and zidovudine.75. The method of claim 66, wherein said patient is undergoingautologous transfusion prior to surgery.
 76. The method of claim 66,wherein said patient is recovering from allogenic bone marrowtransplant.
 77. The method of claim 66, wherein said patient isafflicted with rheumatoid arthritis.
 78. The method of claim 66, whereinsaid dosage regimens are subcutaneous dosage regimens.
 79. A method forobtaining optimized EPO dosage regimens for a desired pharmacokineticresponse in a patient comprising the steps of: (a) selecting one or moredesired pharmacokinetic responses; (b) using apharmacokinetic/pharmacodynamic model to determine EPO dosage regimensthat provide said desired one or more pharmacokinetic responses; and (c)selecting one or more EPO dosage regimens that provide said desiredpharmacokinetic responses.
 80. The method of claim 79, wherein saidpharmacokinetic response comprises of one or more of the groupconsisting of serum EPO levels, bioavailability, and threshold level.81. The method of claim 79, wherein said patient is anemic.
 82. Themethod of claim 81, wherein said anemia comprises EPO concentrationrelated anemia.
 83. The method of claim 82, wherein said anemiacomprises end-stage renal or renal failure related anemia.
 84. Themethod of claim 82, wherein said anemia comprises cancer chemotherapyrelated anemia.
 85. The method of claim 82, wherein said anemiacomprises AIDS drug therapy related anemia.
 86. The method of claim 82,wherein said anemia comprises drug related anemia.
 87. The method ofclaim 86, wherein said drug include cisplatin and zidovudine.
 88. Themethod of claim 79, wherein said patient is undergoing autologoustransfusion prior to surgery.
 89. The method of claim 79, wherein saidpatient is recovering from allogenic bone marrow transplant.
 90. Themethod of claim 79, wherein said patient is afflicted with rheumatoidarthritis.
 91. The method of claim 79, wherein said dosage regimens aresubcutaneous dosage regimens.
 92. A computer program for obtainingoptimized EPO dosage regimens for a desired pharmacokinetic response ina patient comprising: (a) computer code that describes apharmacokinetic/pharmacodynamic model for EPO, said code providing forselection of one or more desired pharmacokinetic responses and the useof said pharmacokinetic/pharmacodynamic model to determine one or moreEPO dosage regimens that provide said desired one or morepharmacokinetic responses; and (b) computer readable medium that storessaid computer code.
 93. The method of claim 92, wherein saidpharmacokinetic response comprises of one or more of the groupconsisting of serum EPO levels, bioavailability, and threshold level.94. The method of claim 92, wherein said patient is anemic.
 95. Themethod of claim 94, wherein said anemia comprises EPO concentrationrelated anemia.
 96. The method of claim 95, wherein said anemiacomprises end-stage renal or renal failure related anemia.
 97. Themethod of claim 95, wherein said anemia comprises cancer chemotherapyrelated anemia.
 98. The method of claim 95, wherein said anemiacomprises AIDS drug therapy related anemia.
 99. The method of claim 95,wherein said anemia comprises drug related anemia.
 100. The method ofclaim 99, wherein said drug include cisplatin and zidovudine.
 101. Themethod of claim 92, wherein said patient is undergoing autologoustransfusion prior to surgery.
 102. The method of claim 92, wherein saidpatient is recovering from allogenic bone marrow transplant.
 103. Themethod of claim 92, wherein said patient is afflicted with rheumatoidarthritis.
 104. The method of claim 92, wherein said dosage regimens aresubcutaneous dosage regimens.
 105. A computer program for obtainingoptimized EPO dosage regimens for a desired pharmacokinetic response ina patient comprising: (a) computer code that describes apharmacokinetic/pharmacodynamic model for EPO, said code providing foruser selection of one or more EPO dosage regimens and the use of saidpharmacokinetic/pharmacodynamic model to determine a pharmacokineticresponse for said one or more EPO dosage regimens; and (b) computerreadable medium that stores said computer code.
 106. The method of claim105, wherein said pharmacokinetic response comprises of one or more ofthe group consisting of serum EPO levels, bioavailability, and thresholdlevel.
 107. The method of claim 105, wherein said patient is anemic.108. The method of claim 107, wherein said anemia comprises EPOconcentration related anemia.
 109. The method of claim 108, wherein saidanemia comprises end-stage renal or renal failure related anemia. 110.The method of claim 108, wherein said anemia comprises cancerchemotherapy related anemia.
 111. The method of claim 108, wherein saidanemia comprises AIDS drug therapy related anemia.
 112. The method ofclaim 108, wherein said anemia comprises drug related anemia.
 113. Themethod of claim 112, wherein said drug include cisplatin and zidovudine.114. The method of claim 105, wherein said patient is undergoingautologous transfusion prior to surgery.
 115. The method of claim 105,wherein said patient is recovering from allogenic bone marrowtransplant.
 116. The method of claim 105, wherein said patient isafflicted with rheumatoid arthritis.
 117. The method of claim 105,wherein said dosage regimens are subcutaneous dosage regimens.
 118. Amethod for creating a pharmacokinetic model for subcutaneous EPOadministration in patients comprising the steps of: (a) obtainingpharmacokinetic data from patients; (b) choosing an equation based onsaid data; and (c) fitting said pharmacokinetic data to said equation.119. A method for creating a pharmacodynamic model for subcutaneous EPOadministration in patients comprising the steps of: (a) normalizingserum EPO concentrations; (b) obtaining pharmacodynamic data; (c)choosing a pharmacodynamic model; (d) obtaining equation based on saidmodel; and (e) fitting pharmacodynamic data to said equation.
 120. Themethod of claim 118, wherein said obtaining pharmacokinetic datacomprises: (a) normalizing serum EPO concentration values from saidpharmacokinetic data; and (b) creating serum EPO versus time profilesbased on said normalized data.
 121. The method of claim 120, whereinsaid normalizing step comprises: (a) obtaining baseline serum EPOconcentration values from said pharmacokinetic data by averaging predoseserum EPO concentration values at plurality of time points; (b)obtaining serum EPO concentration values following subcutaneous EPOadministration; (c) obtaining normalized serum EPO concentration valuesby subtracting predose EPO concentration values from serum EPOconcentration values; and (d) calculating mean normalized serum EPOconcentration values at each time point.
 122. The method of claim 118,wherein said pharmacokinetic equation comprises the Michaelis-Mentenequation.
 123. The method of claim 118, wherein said fitting stepcomprises obtaining estimates of pharmacokinetic parameters utilizingleast-squares by Maximum Likelihood method and extended least squaresmodel.
 124. The method of claim 123, wherein said parameters areselected from the group consisting of Vmax, Km, Vd, Ka, Fr, τ (lowerdoses), and τ (higher dose).
 125. The method of claim 123, wherein saidfitting step comprises utilizing ADAPT II software.
 126. A method forcalculating the bioavailability of EPO following subcutaneousadministration comprises the steps of: (a) obtaining pharmacokineticdata; (b) calculating AUC; (c) normalizing AUC to dose; and (d) derivingan equation to represent said bioavailability of EPO by performing alinear regression of said pharmacokinetic data.
 127. The method of claim119, wherein said normalizing step comprises: (a) obtaining baselineserum EPO concentration (C_(bs)) for each dose group by averagingpredose serum EPO concentration values at plurality of time points foreach dose group; and (b) adjusting C_(bs) by adding C_(bs) to serum EPOconcentration predicted by pharmacokinetic model wherein said adjustedCbs may be used as a forcing function for pharmacodynamic analysis. 128.The method of claim 119, wherein said obtaining pharmacodynamic datastep comprises: (a) determining mean predose precursor cell number; (b)determining mean predose reticolucyte number; (c) determining meanpredose RBC number; (d) determining mean predose hemoglobinconcentration; (e) obtaining mean reticulocyte versus time profilesaccording to EPO dose; (f) obtaining mean RBC versus time profilesaccording to EPO dose; and (g) obtaining mean hemoglobin versus timeprofiles according to EPO dose.
 129. The method of claim 119, whereinsaid pharmacodynamic model comprises a cell production and cell lossmodel.
 130. The method of claim 119, wherein said fitting step comprisesobtaining parameters utilizing least squares by Maximum Likelihoodmethod and extended least squares model.
 131. The method of claim 130,wherein said parameters comprise estimated parameters and fixedparameters.
 132. The method of claim 131, wherein said estimatedparameters comprise Ks, SC₅₀, and TP.
 133. The method of claim 131,wherein said fixed parameters comprise R_(L), RBC_(L), Hb, andthreshold.
 134. The method of claim 130, wherein said fitting stepcomprises utilizing ADAPT II software.
 135. A method for predicting apharmacodynamic response in a patient to subcutaneous EPO administrationcomprising the steps of: (a) selecting EPO dose and dosage regimens; and(b) determining said pharmacodynamic response based on said dose anddosage regimens.
 136. The method of claim 135, wherein saidpharmacodynamic response comprises of one or more of the groupconsisting of reticulocyte number, RBC number, and hemoglobin level.137. The method of claim 135, wherein said patient is anemic.
 138. Themethod of claim 137, wherein said anemia comprises EPO concentrationrelated anemia.
 139. The method of claim 138, wherein said anemiacomprises end-stage renal or renal failure related anemia.
 140. Themethod of claim 138, wherein said anemia comprises cancer chemotherapyrelated anemia.
 141. The method of claim 138, wherein said anemiacomprises AIDS drug therapy related anemia.
 142. The method of claim138, wherein said anemia comprises drug related anemia.
 143. The methodof claim 142, wherein said drug include cisplatin and zidovudine. 144.The method of claim 135, wherein said patient is undergoing autologoustransfusion prior to surgery.
 145. The method of claim 135, wherein saidpatient is recovering from allogenic bone marrow transplant.
 146. Themethod of claim 135, wherein said patient is afflicted with rheumatoidarthritis.
 147. The method of claim 135, wherein said dosage regimensare subcutaneous dosage regimens.
 148. A method for administering EPOcomprising the steps of: choosing one or more EPO dosage regimens usinga pharmacokinetic/pharmacodynamic model to determine the pharmacodynamicprofile of said one or more EPO regimens; selecting said EPO dosageregimens that provides a desired pharmacodynamic response based on saidpharmacodynamic profile; and administering said EPO dosage regimen to apatient.
 149. The method of claim 148, wherein said EPO dosing regimencomprises administering EPO once a week.
 150. The method of claim 148,wherein said EPO dosing regimen comprises administering EPO twice aweek.
 151. The method of claim 148, wherein said patient is anemic. 152.The method of claim 151, wherein said anemia comprises EPO concentrationrelated anemia.
 153. The method of claim 151, wherein said anemiacomprises end-stage renal or renal failure related anemia.
 154. Themethod of claim 151, wherein said anemia comprises cancer chemotherapyrelated anemia.
 155. The method of claim 151, wherein said anemiacomprises AIDS drug therapy related anemia.
 156. The method of claim151, wherein said anemia comprises drug related anemia.
 157. The methodof claim 156, wherein said drug is selected from the group consisting ofcisplatin and zidovudine.
 158. The method of claim 148, wherein saidpatient is undergoing autologous transfusion prior to surgery.
 159. Themethod of claim 148, wherein said patient is recovering from allogenicbone marrow transplant.
 160. The method of claim 148, wherein saidpatient is afflicted with rheumatoid arthritis.
 161. The method of claim148, wherein said EPO dosage regimens are administered subcutaneously.162. A method of administering EPO comprising the steps of: selectingone or more desired pharmacodynamic responses; using apharmacokinetic/pharmacodynamic model to determine EPO dosage regimenthat provides said desired one or more pharmacodynamic responses;selecting said one ore more EPO dosage regimens that provides saiddesired pharmacodynamic responses; and administering said selected EPOdosage regiment to a patient.
 163. The method of claim 162, wherein saidEPO dosage regimen comprises administering EPO once a week.
 164. Themethod of claim 162, wherein said EPO dosage regiment comprisesadministering EPO once every weeks.
 165. The method of claim 162,wherein said pharmacodynamic responses are selected from the groupsconsisting of reticulocyte number, RBC number, and hemoglobin level.166. The method of claim 162, wherein said patient is anemic.
 167. Themethod of claim 162, wherein said anemia comprises EPO concentrationrelated anemia.
 168. The method of claim 162, wherein said anemiacomprises end-stage renal or renal failure related anemia.
 169. Themethod of claim 162, wherein said anemia comprises cancer chemotherapyrelated anemia.
 170. The method of claim 162, wherein said anemiacomprises AIDS drug therapy related anemia.
 171. The method of claim162, wherein said anemia comprises drug related anemia.
 172. The methodof claim 171, wherein said drug is selected from the group consisting ofcisplatin and zidovudine.
 173. The method of claim 162, wherein saidpatient is undergoing autologous transfusion prior to surgery.
 174. Themethod of claim 162, wherein said patient is recovering from allogenicbone marrow transplant.
 175. The method of claim 162, wherein saidpatient is afflicted with rheumatoid arthritis.
 176. The method of claim162, wherein said EPO dosage regimens are administered subcutaneously.177. A method for administering EPO to a patient comprising the step of:administering said EPO on a once-weekly basis.
 178. The method of claim177, wherein said administering comprises a dose 40,000 IU of said EPO.179. A method for administering EPO to a patient comprising the step of:administering said EPO on a once every two week basis.
 180. The methodof claim 179, wherein said administering comprises a dose selected fromthe group consisting of 80,000 IU/kg, 100,000 IU/kg, and 120,000 IU/kg.181. A method for enhancing the production of mature red blood cellsfrom young red blood cells in a patient comprising the step of:administering EPO to said patient so that said young red blood cells areinduced to become mature red blood cells.
 182. A method for maintainingan enhanced level of red blood cells in a patient comprising the stepof: administering a first dose of EPO followed by a second dose of EPOto said patient, wherein said second dose of EPO is administered to saidpatient at a time after said first dose that coincides with theproduction of reticulocytes resulting from said first dose of EPO. 183.The method of claim 182, wherein said second dose of EPO is administeredto said patient between six and twelve days after said first dose. 184.The method of claim 182, wherein said second dose of EPO is administeredto said patient between six and ten days after said first dose.
 185. Themethod of claim 182, wherein said second dose of EPO is administered tosaid patient seven days after said first dose.
 186. A business methodcomprising the step of: providing to a consumer an EPO dosing regimenthat is a first dose of EPO followed by a second dose of EPO to apatient, wherein said second dose of EPO is administered to said patientat a time after said first dose that coincides with the production ofreticulocytes resulting from said first dose of EPO.
 187. The method ofclaim 186, wherein said EPO dose regimen comprises dosing one time perweek with an effective amount of EPO.
 188. The method of claim 187,wherein said effective amount of EPO comprises 40,000 IU/kg.
 189. Themethod of claim 186, wherein said EPO dose regimen comprises dosing onceevery two weeks with an effective amount of EPO.
 190. The method ofclaim 189, wherein said effective amount of EPO is selected from thegroup consisting of 80,000 IU/kg, 100,000 IU/kg, and 120,000 IU/kg. 191.A business method comprising the step of: providing to a patient an EPOdosing regimen that is a first dose of EPO followed by a second dose ofEPO to a patient, wherein said second dose of EPO is administered tosaid patient at a time after said first dose that coincides with theproduction of reticulocytes resulting from said first dose of EPO. 192.The method of claim 191, wherein said EPO dose regimen comprises dosingone time per week with an effective amount of EPO.
 193. The method ofclaim 192, wherein said effective amount of EPO comprises 40,000 IU/kg.194. The method of claim 191, wherein said EPO dose regimen comprisesdosing once every two weeks with an effective amount of EPO.
 195. Themethod of claim 194, wherein said effective amount of EPO is selectedfrom the group consisting of 80,000 IU/kg, 100,000 IU/kg, and 120,000IU/kg.
 196. A business method comprising the step of: providing a dosingregimen of EPO to a user or patient.
 197. The method of claim 196,wherein said dosing regimen is once weekly.
 198. The method of claim196, wherein said dosing regimen is once every two weeks.
 199. Themethod of claim 196 further comprising the step of: providing EPO inconjunction with said providing a dosing regimen of EPO to a user orpatient.
 200. The method of claim 196, wherein said providing stepcomprises selling.
 201. The method of claim 199, wherein said providingstep comprises selling.
 202. The method of claim 196, wherein saidproviding step is performed through the use of a computer system. 203.The method of claim 199, wherein said providing step is performedthrough the use of a computer system.